A method for detecting tumor redox microenvironment using extremely low field magnetic resonance

Abstract

The present invention relates to a method for detecting the tumor redox microenvironment using extremely low-field magnetic resonance imaging (ELR). The method comprises sequentially adding water, polyethylene glycol, and gadolinium nitrate to cysteine hydrochloride for a hydrothermal reaction to produce a fourth mixed system; adding equal volumes of a simulated tumor microenvironment and water to each of the experimental and control groups, respectively; and measuring the relaxation times Ta and Tb of the experimental and control groups in an ELR system, and comparing the difference between Ta and Tb. This method converts difficult-to-detect differences in the tumor redox microenvironment into differences in magnetic relaxation times, quantifying these differences. The method also exhibits excellent detection sensitivity and has promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomarker detection, and particularly relates to a method for detecting tumor redox microenvironment using extremely low field magnetic resonance. Background Art

[0002] Common tumor microenvironment detection technologies focus on differences in pH, metabolic environment, and proliferation behavior within the tumor microenvironment. Tumor microenvironment detection based on differences in metabolic behavior has advantages in studying the mechanisms of tumorigenesis and detecting tumors in the very early stages. Among the differences in tumor metabolic behavior, one important marker that can be detected is redox markers, such as oxygen free radicals, homocysteine, and oxidized glutathione. A variety of fluorescence-based detection methods have been applied to the detection of these markers, but fluorescence-based detection technologies have disadvantages such as low signal penetration depth and susceptibility to interference from background fluorescence signals in the sample.

[0003] Compared to fluorescence-based detection techniques, magnetic resonance (MRI) detection based on magnetic signals offers advantages such as deeper signal penetration and reduced susceptibility to background signal interference from the sample (due to the low magnetic susceptibility of biological media). In MRI, magnetic relaxation time can be used to analyze differences between substances, but changes in the tumor redox microenvironment have little effect on magnetic relaxation time. Therefore, how to convert differences in the tumor redox microenvironment into differences in magnetic relaxation time and detect them is a key challenge in the field of magnetic detection of the tumor microenvironment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting the tumor redox microenvironment using extremely low-field magnetic resonance. This method converts difficult-to-detect differences in the tumor redox microenvironment into differences in magnetic relaxation times, quantifies the differences, and has excellent detection sensitivity and good application prospects.

[0005] The present invention provides a method for detecting tumor redox microenvironment using extremely low field magnetic resonance, comprising the following steps: S1, dissolving cysteine hydrochloride in water to obtain a first mixed system;

[0006] S2, subjecting the first mixed system to a hydrothermal reaction, and obtaining a second mixed system through filtration and dialysis;

[0007] S3, adding polyethylene glycol to the second mixed system, and obtaining a third mixed system through hydrothermal reaction, filtration, and dialysis;

[0008] S4, adding gadolinium nitrate to the third mixed system, performing hydrothermal reaction, filtering, and dialysis to obtain a fourth mixed system;

[0009] S5. Add equal volumes of simulated tumor microenvironment and water to the fourth mixed system, which are recorded as the experimental group and the control group;

[0010] S6. Detect the relaxation times Ta and Tb of the experimental group and the control group in an extremely low field magnetic resonance system, and compare the differences between Ta and Tb.

[0011] The concentration of the first mixed system in step S1, i.e., the cysteine hydrochloride solution, is 10 μg / mL-10 mg / mL.

[0012] The degree of polymerization of polyethylene glycol in step S3 is 8-16, and the amount added is 1 μmol-1 mmol.

[0013] The molar ratio of gadolinium nitrate to polyethylene glycol in step S4 is 1:1.

[0014] The simulated tumor microenvironment in step S5 is simulated by a hydrogen peroxide solution having a concentration of 2 nmol / L-2 mmol / L; the volume ratio of the total volume of the simulated tumor microenvironment and water to the volume of the fourth mixed system is 1:1.

[0015] The hydrothermal reaction temperature in steps S2, S3, and S4 is 120-200° C., and the hydrothermal reaction time is 6-24 hours.

[0016] The filtration in steps S2, S3 and S4 uses a filter membrane with a pore size of 0.2 to 2 μm.

[0017] The dialysis in steps S2, S3 and S4 uses a dialysis bag with a molecular cut-off of 500 to 5000 Da.

[0018] The main magnetic field strength of the extremely low field magnetic resonance system in step S6 is 25-250 μT.

[0019] Comparing the difference between Ta and Tb in step S6 is to convert the difference in tumor redox microenvironment into the difference in magnetic relaxation time.

[0020] Paramagnetic gadolinium ions dissolved in water can alter the magnetic relaxation time of water, but gadolinium ions are toxic and cannot match the redox potential of the tumor microenvironment. Therefore, a binding structure is needed to stabilize the gadolinium ions and further transmit changes in the tumor redox microenvironment to the gadolinium ions, thereby changing the magnetic relaxation time of water in the magnetic resonance imaging system. Graphene quantum dots are carbon nanostructures with rich surface functional groups that can be linked to a variety of molecules, serving as a structure for binding gadolinium ions and transmitting changes in the tumor redox microenvironment.

[0021] Among various magnetic resonance systems, the extremely low field magnetic resonance system operating in an extremely low magnetic field (main magnetic field of the order of μT) has a significant advantage in high magnetic molecular relaxation rate compared with the high-field magnetic resonance system currently used in clinical practice. When the tumor redox microenvironment is the same, the difference in magnetic relaxation time measured in the extremely low field magnetic resonance system and the control group is greater than the difference in magnetic relaxation time measured in the high-field magnetic resonance system.

[0022] Beneficial effects

[0023] The present invention converts difficult-to-detect differences in tumor redox microenvironment into differences in magnetic relaxation time, quantifies the differences, and has excellent detection sensitivity and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the process of the present invention;

[0025] Figure 2 is the relaxation time of Sample 1 and Sample 2 in Example 1 measured in an extremely low field magnetic resonance system;

[0026] Figure 3 is Ta measured under an extremely low field magnetic resonance system when the fourth mixed system obtained in Example 2 reacts with a 2 mmol / L hydrogen peroxide solution for different times;

[0027] Figure 4 Ta is the Ta measured under an extremely low field magnetic resonance system after the fourth mixed system obtained in Example 2 reacts with simulated tumor microenvironments of different concentrations;

[0028] Figure 5 Ta is the Ta measured under an extremely low field magnetic resonance system when the fourth mixed system obtained in Example 3 reacts with hydrogen peroxide solutions of different concentrations. DETAILED DESCRIPTION

[0029] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0030] Example 1

[0031] Combine Figure 1 This embodiment provides a method for detecting tumor redox microenvironment using extremely low field magnetic resonance imaging, comprising the following steps:

[0032] S1, providing cysteine hydrochloride;

[0033] S2. Dissolving cysteine hydrochloride in water to obtain a first mixed system having a volume of 10 mL and a cysteine hydrochloride concentration of 1 mg / mL;

[0034] S3. The first mixed system is subjected to hydrothermal reaction, filtration, dialysis and other operations to obtain a second mixed system. The hydrothermal reaction temperature is 150° C., the time is 12 h, the pore size of the filter membrane used for filtration is 0.2 μm, and the molecular cutoff of the dialysis bag used for dialysis is 3000 Da.

[0035] S4. Add polyethylene glycol (PEG) with a degree of polymerization of 12 and an amount of 0.1 mmol to the second mixed system. Perform hydrothermal reaction, filtration, dialysis, and other operations on the mixed system to obtain a third mixed system. The hydrothermal reaction, filtration, dialysis, and other operating conditions are the same as those in step S3 of this embodiment.

[0036] S5. Add gadolinium nitrate to the third mixed system, with the molar ratio of gadolinium nitrate to polyethylene glycol added in step S4 being 1:1. Perform hydrothermal reaction, filtration, dialysis, and other operations on the mixed system to obtain a fourth mixed system. The hydrothermal reaction, filtration, dialysis, and other operating conditions are the same as those in step S3 of this embodiment.

[0037] S6. Add equal volumes of simulated tumor microenvironment and water to equal volumes of the fourth mixed system, respectively, to form the experimental group and the control group. The simulated tumor microenvironment is a hydrogen peroxide solution with a concentration of 2 nmol / L.

[0038] S7. The relaxation times Ta and Tb of the experimental group and the control group were measured in an extremely low field magnetic resonance system with a main magnetic field strength of 100 μT.

[0039] like Figure 2 As shown, sample 1 is the relaxation time Ta measured by the experimental group in the extremely low field magnetic resonance system, which is 231.28±5.77ms, and sample 2 is the relaxation time Tb measured by the control group in the extremely low field magnetic resonance system, which is 406.12±8.03ms. There is a significant difference between Ta and Tb, indicating that the present invention can convert the change of the redox environment into the change of the relaxation time under the extremely low field magnetic resonance system, thereby realizing the quantification of the redox environment.

[0040] Example 2

[0041] Combine Figure 1 This embodiment provides a method for detecting tumor redox microenvironment using extremely low field magnetic resonance imaging, comprising the following steps:

[0042] S1, providing cysteine hydrochloride;

[0043] S2. Dissolving cysteine hydrochloride in water to obtain a first mixed system having a volume of 10 mL and a cysteine hydrochloride concentration of 10 μg / mL;

[0044] S3. The first mixed system is subjected to hydrothermal reaction, filtration, dialysis and other operations to obtain a second mixed system. The hydrothermal reaction temperature is 200°C, the time is 6 hours, the pore size of the filter membrane used for filtration is 2 μm, and the molecular cutoff of the dialysis bag used for dialysis is 5000 Da.

[0045] S4. Add polyethylene glycol (PEG) with a degree of polymerization of 8 and an amount of 1 mmol to the second mixed system. Perform hydrothermal reaction, filtration, dialysis, and other operations on the mixed system to obtain a third mixed system. The hydrothermal reaction, filtration, dialysis, and other operating conditions are the same as those in step S3 of this embodiment.

[0046] S5. Add gadolinium nitrate to the third mixed system, with the molar ratio of gadolinium nitrate to polyethylene glycol added in step S4 being 1:1. Perform hydrothermal reaction, filtration, dialysis, and other operations on the mixed system to obtain a fourth mixed system. The hydrothermal reaction, filtration, dialysis, and other operating conditions are the same as those in step S3 of this embodiment.

[0047] S6. Add equal volumes of simulated tumor microenvironment and water to equal volumes of the fourth mixed system, respectively, and record them as experimental group and control group. The simulated tumor microenvironment is hydrogen peroxide solution with concentrations of 2 nmol / L, 20 nmol / L, 0.2 mmol / L, and 2 mmol / L.

[0048] S7. The relaxation times Ta and Tb of the experimental group and the control group were measured in an extremely low field magnetic resonance system, where the main magnetic field strength of the extremely low field magnetic resonance system was 25 μT.

[0049] Fourth, the reaction time between the hybrid system and the simulated tumor microenvironment will affect the difference between Ta and Tb measured in the ultra-low field magnetic resonance system. Figure 3 The Ta values measured by an ultra-low field magnetic resonance system when the fourth mixed system obtained in this example reacted with a 2 mmol / L hydrogen peroxide solution for different times are given. Figure 3 As shown, Ta at time 0 is equal to Tb (577.19±8.44ms). As the reaction time increases, Ta gradually shortens and stabilizes at around 230ms, indicating that the reaction is sufficient 50 minutes after the fourth mixed system reacts in the hydrogen peroxide solution, and the difference between Ta and Tb is most significant at this time.

[0050] The Ta measured in the ultra-low field magnetic resonance system after the same fourth mixed system reacts with different concentrations of simulated tumor microenvironment will also be different, such as Figure 4As shown in the figure, with the increase of hydrogen peroxide concentration, the difference between Ta and Tb gradually increases, indicating that the relaxation time of the fourth mixed system changes greatly in a strong oxidizing environment. Further, a linear fit is made with Ta as the ordinate and the logarithm of hydrogen peroxide concentration as the abscissa, and we can get y=–93.60x+540.73, and the fitting R 2 The value is 0.99996, indicating high data linearity, which can be used to achieve quantitative detection.

[0051] Example 3

[0052] Combine Figure 1 This embodiment provides a method for detecting tumor redox microenvironment using extremely low field magnetic resonance imaging, comprising the following steps:

[0053] S1, providing cysteine hydrochloride;

[0054] S2. Dissolving cysteine hydrochloride in water to obtain a first mixed system having a volume of 10 mL and a cysteine hydrochloride concentration of 10 mg / mL;

[0055] S3. The first mixed system is subjected to hydrothermal reaction, filtration, dialysis and other operations to obtain a second mixed system. The hydrothermal reaction temperature is 120° C., the time is 24 h, the pore size of the filter membrane used for filtration is 1 μm, and the molecular cutoff of the dialysis bag used for dialysis is 500 Da.

[0056] S4. Add polyethylene glycol (PEG) with a degree of polymerization of 16 and an amount of 1 μmol to the second mixed system. Perform hydrothermal reaction, filtration, dialysis, and other operations on the mixed system to obtain a third mixed system. The hydrothermal reaction, filtration, dialysis, and other operating conditions are the same as those in step S3 of this embodiment.

[0057] S5. Add gadolinium nitrate to the third mixed system, with the molar ratio of gadolinium nitrate to polyethylene glycol added in step S4 being 1:1. Perform hydrothermal reaction, filtration, dialysis, and other operations on the mixed system to obtain a fourth mixed system. The hydrothermal reaction, filtration, dialysis, and other operating conditions are the same as those in step S3 of this embodiment.

[0058] S6. Equal volumes of simulated tumor microenvironment and water were added to equal volumes of the fourth mixed system, respectively, and recorded as the experimental group and the control group. The simulated tumor microenvironment was a hydrogen peroxide solution with concentrations of 1 pmol / L, 10 pmol / L, 0.1 nmol / L, 1 nmol / L, 10 nmol / L, 0.1 mmol / L, and 1 mmol / L.

[0059] S7. The relaxation times Ta and Tb of the experimental group and the control group were measured in an extremely low field magnetic resonance system with a main magnetic field strength of 250 μT.

[0060] like Figure 5As shown in FIG, the Ta comparison of the fourth mixed system obtained in this embodiment when detecting various concentrations of hydrogen peroxide is shown. The Ta corresponding to zero concentration is equal to Tb (671.41±10.87ms). Figure 5 It can be seen that as the hydrogen peroxide concentration increases, Ta gradually decreases, that is, the difference between Ta and Tb increases. When the hydrogen peroxide concentration is 1 pmol / L, Ta is 657.17±9.70 ms, which is still significantly different from Tb, indicating that the detection sensitivity of the fourth mixed system obtained in this example is better than 1 pmol / L, which is better than the currently known hydrogen peroxide detection method (the detection limit in the reported paper is about 2.8 nmol / L, Analyst, 2019, 144(13): 4006-4012.).

Claims

1. A method for detecting the tumor redox microenvironment using extremely low field magnetic resonance imaging, comprising the following steps: S1. Dissolving cysteine hydrochloride in water to obtain a first mixed system, wherein the concentration of the first mixed system, i.e., the cysteine hydrochloride solution, is 10 μg / mL to 10 mg / mL; S2, subjecting the first mixed system to a hydrothermal reaction, and obtaining a second mixed system through filtration and dialysis; S3, adding polyethylene glycol to the second mixed system, and obtaining a third mixed system through hydrothermal reaction, filtration, and dialysis; wherein the amount of polyethylene glycol added is 1 μmol-1 mmol; S4. Adding gadolinium nitrate to the third mixed system, performing hydrothermal reaction, filtering, and dialysis to obtain a fourth mixed system; wherein the molar ratio of the gadolinium nitrate to the polyethylene glycol is 1:1; S5. Add equal volumes of simulated tumor microenvironment and water to two equal volumes of the fourth mixed system, respectively, to form an experimental group and a control group; the simulated tumor microenvironment is simulated by a hydrogen peroxide solution having a concentration of 2 nmol / L to 2 mmol / L; S6. Detecting the relaxation times Ta and Tb of the experimental group and the control group in an extremely low field magnetic resonance system, and comparing the difference between Ta and Tb; the main magnetic field strength of the extremely low field magnetic resonance system is 25 to 250 μT.

2. The method according to claim 1, wherein: The degree of polymerization of the polyethylene glycol in step S3 is 8-16.

3. The method according to claim 1, wherein: In step S5, the ratio of the total volume of the simulated tumor microenvironment and water to the total volume of the two equal parts of the fourth mixed system is 1:

1.

4. The method according to claim 1, wherein: The hydrothermal reaction temperature in steps S2, S3, and S4 is 120-200° C., and the hydrothermal reaction time is 6-24 hours.

5. The method according to claim 1, wherein: The filtration in steps S2, S3 and S4 uses a filter membrane with a pore size of 0.2 to 2 μm.

6. The method according to claim 1, wherein: The dialysis in steps S2, S3 and S4 uses a dialysis bag with a molecular cut-off of 500 to 5000 Da.

7. The method according to claim 1, wherein: Comparing the difference between Ta and Tb in step S6 is to convert the difference in tumor redox microenvironment into the difference in magnetic relaxation time.

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