Preparation method and application of low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme catalysis cascade signal amplification

Abstract

The invention discloses a preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme catalysis cascade signal amplification. The preparation method is characterized by comprising the following steps: 1) preparing a recognition unit Fe3O4 (at) DNA2 / Apt; (2) an initiator GDA (at) GOXamp; preparing DNA1 (deoxyribonucleic acid 1); (3) preparing a signal unit CaO2 (at) MnO2; (4) preparing the LF-NMR biosensor; determining the intensity of corresponding low-field nuclear magnetic resonance signals T2 under the condition of CTC with different concentrations by adopting a low-field nuclear magnetic resonance instrument, establishing a quantitative relationship between the CTC concentration and a T2 signal change value (delta T2), and determining the concentration of CTC in an unknown sample according to the quantitative relationship; the method has the advantages of high sensitivity, high selectivity and high expandability.

Description

Technical Field

[0001] The present invention relates to a low-field nuclear magnetic resonance sensor, and in particular to a preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification. Background Art

[0002] Tumor metastasis is the main cause of death in cancer patients. Circulating tumor cells (CTCs), which are tumor cells that shed from primary tumors and enter the circulatory system, play a key role in tumor metastasis and have become an important biomarker for early diagnosis of cancer, patient staging, and efficacy evaluation. However, the concentration of CTCs in whole blood samples is very low, and their high-sensitivity detection still faces huge technical challenges. Existing methods such as flow cytometry, CellSearch system, and confocal microscopy are expensive and their widespread application is limited. New CTC sensing detection methods have also been developed, such as electrochemistry, electrochemiluminescence, photoelectrochemistry, fluorescence, colorimetry, surface-enhanced Raman, etc. These methods show certain advantages in sensitivity, cost, and operation, but usually require cumbersome sample pretreatment to eliminate signal interference caused by complex sample matrices.

[0003] In contrast, magnetic materials rarely appear in biological sample matrices, so magnetic sensing methods have unique advantages in anti-interference. In addition, the magnetic sensing method based on low-field nuclear magnetic resonance (LF-NMR) technology has simple instruments, convenient operation, small sample volume, and low analysis cost. It has been widely used to detect pathogens, pesticide residues, and heavy metal ions, but there has been no report on its use for CTC detection. The main reason is that the number of CTCs in peripheral blood is extremely small and the interference is extremely large, with only one CTC per million normal blood cells. Because the applied magnetic field intensity is low, sensitivity has always been the "Achilles' heel" of LF-NMR detection, and the signal amplification strategy must be carefully designed to achieve high-sensitivity detection of CTCs.

[0004] The present invention is based on the recognition unit Fe 3 O 4 @DNA2 / Apt, initiator GDA@GOX&DNA1 and signal unit CaO 2 @MnO 2 In this study, a target-triggered, cyclically amplified LF-NMR sensing platform was constructed for the detection of CTCs. When the target CTC is present, the magnetic hybrid disintegrates and hybridizes with the initiator GDA@GOX&DNA1, triggering the following cyclic reactions: (1) Glucose oxidase (GOX) catalyzes the conversion of substrate glucose and oxygen (O 2 ) to produce gluconic acid and hydrogen peroxide (H 2 O 2 );(2) Generated H 2 O2 With CaO 2 @MnO 2 MnO 2 Reaction occurs to generate Mn 2+ and O 2 ; (3) The production of gluconic acid lowers the pH of the system, CaO 2 Acid hydrolysis continues to produce H 2 O 2 ; (4) O produced by the above reaction 2 and H 2 O 2 Continue to participate in reactions (1) and (2), forming an avalanche cycle amplification effect. The large amount of Mn released 2+ The generation of magnetic signals can achieve highly sensitive detection of CTCs. Currently, there are no reports on the detection of CTCs using magnetic sensing methods based on LF-NMR. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification with high sensitivity and convenient operation.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification. This method is not for the purpose of diagnosis or treatment, and includes the following steps:

[0007] (1) Identification unit Fe 3 O 4 Preparation of @DNA2 / Apt

[0008] Take 2.5-7.5 mg Fe 3 O 4 -NH 2 Add 2.5-7.5 mL of 5.0 wt% glutaraldehyde aqueous solution, stir at room temperature for 1-4 h and wash with water, then redisperse in 2.9-6.9 mL PBS, add 50-200 μL of 50 μmol / L DNA2 solution, incubate at 37°C with slow shaking for 2-4 h, and then separate and wash with a magnetic separator. Add 50-150 μL of 5 wt% BSA to block nonspecific sites, redisperse in 3.5-6.0 mL PBS, add 50-150 μL of 100 μmol / L CTC aptamer (Apt) solution, incubate for 1-3 h, wash with a magnetic separator, and redisperse in 3.5-6.0 mL PBS to obtain Fe 3 O 4 @DNA2 / Apt dispersion.

[0009] (2) Preparation of initiator GDA@GOX&DNA1

[0010] 3.5-6.0 mL 1.0 mg / mL glucose oxidase (GOX) solution and 0.05-0.15 mL 50 μmol / L DNA1 solution were added to 4.0-6.0 mL GDA dispersion, and incubated at 4°C for 8-12 h. GOX and DNA1 were assembled on the GDA surface through the reaction of amino groups with catechol groups. 50-150 μL 5 wt% BSA was added to block non-specific sites, washed by centrifugation, and dispersed in 3.5-6.0 mL pH = 7.4 PBS solution to obtain GDA@GOX&DNA1 dispersion.

[0011] (3) Signal unit CaO 2 @MnO 2 Preparation

[0012] 1.0~3.0mg CaO 2 Disperse in 1.0-3.0 mL of anhydrous ethanol, add 50-150 μL of 10 mmol / mL morpholineethanesulfonic acid (MES), and continue ultrasonication for 20-40 min. Then, centrifuge, wash, and redisperse in 1.0-3.0 mL of aqueous solution, add 100-300 μL of 3 mg / mL KMnO 4 The solution was ultrasonicated for 20 to 40 minutes. Finally, it was centrifuged, washed, and redispersed in 1.0 to 3.0 mL of PBS solution to obtain CaO. 2 @MnO 2 Dispersion.

[0013] (4) Preparation of LF-NMR biosensor

[0014] Add 25-75 μL Fe 3 O 4 @DNA2 / Apt dispersion and 25-75 μL GDA@GOX&DNA1 dispersion were added to 0.5-1.5 mL of CTC-containing sample solution and incubated at 37°C for 20-40 min. Magnetic separation, washing and dispersion in 250-750 μL 50 mmol / L pH=6.5 PBS solution were performed, and then 25-75 μL universal nuclease solution (25 U / μL) was added and incubated at 37°C for 15-30 min to release Fe bound to GDA@GOX. 3 O 4 After magnetic separation, take 250-750 μL of the supernatant and add 25-75 μL of 5 mmol / L glucose solution and 25-75 μL of CaO 2 @MnO 2The dispersion was incubated for 20-40 min. After filtration, 250-750 μL of the clear solution was taken for LF-NMR testing.

[0015] Furthermore, the synthesis method of GDA described in step (2) is as follows: 8-12 mg GO is dispersed in 20-30 mL pH = 8.5 0.01 mol / L Tris-HCl solution, and after ultrasonication for 20-40 min, 4-6 mL 1 mg / mL dopamine hydrochloride is added, and stirred at room temperature for 10-14 h to obtain GO (GDA) with polydopamine assembled on the surface. After centrifugal washing and redispersion in 8-12 mL 10 mmol / L pH = 7.4 PBS solution, a GDA dispersion is obtained.

[0016] Further, the CaO in step (3) 2 The synthesis method is as follows: add 4-8 mL 0.1 g / mL CaCl 2 Add 2-4 mL 1 mol / L ammonia and 20-30 mL polyethylene glycol 200 to the solution, then add 4-6 mL 30% H 2 O 2 , continue stirring for 1 to 3 hours. Add 0.1 mol / L NaOH solution to adjust the pH to 10.5 to 12.5, centrifuge and wash, and dry at 80℃ to obtain CaO 2 .

[0017] 2. A method for detecting circulating tumor cells using the above-mentioned low-field nuclear magnetic resonance sensor, which is not intended for diagnosis or treatment, and comprises the following steps: determining the T corresponding to different CTC concentrations at 35°C; 2 Strength, according to T 2 Signal change value (ΔT 2 ) to obtain the concentration of CTC in the test solution.

[0018] Furthermore, the T 2 The signal change value is calculated according to the following formula: ΔT 2 =T 2 (negative)-T 2 (positive), T 2 (negative) is the average T when there are no CTCs in the sample 2 , T 2 (positive) is the average T when CTCs are present in the sample 2 .

[0019] Invention principle: The principle of low-field nuclear magnetic resonance sensor is as follows Figure 1 Taking circulating tumor cells A549 as an example, when A549 is present, the recognition unit Fe3 O 4 @DNA2 / Apt’s aptamer Apt specifically recognizes A549. At this time, the magnetic hybrid Fe 3 O 4 The single-stranded DNA2 on @DNA2 hybridizes with the DNA1 on the initiator GDA@GOX&DNA1 to obtain GDA@GOX&DNA1-DNA2@Fe 3 O 4 It was quickly collected by magnetic force. Then GDA@GOX&DNA1-DNA2@Fe 3 O 4 DNA lysis and magnetic separation to remove Fe 3 O 4 Then, glucose and the signal unit CaO were added to GDA@GOX. 2 @MnO 2 The cascade autocatalytic reaction driven by glucose oxidase on GDA@GOX continuously generates Mn 2+ , producing a stable NMR signal. In this system, the more A549 there is, the more GDA@GOX is magnetically collected, and the Mn 2+ The higher the concentration, the higher the ΔT 2 In this way, quantitative detection of circulating tumor cell A549 is achieved.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] (1) High sensitivity: The signal response intensity of LF-NMR detection is effectively improved through the synergistic effect of enzyme-catalyzed signal amplification and acid-sensitive nanomaterials.

[0022] (2) Strong selectivity: The use of specific aptamers and magnetic separation technology can effectively eliminate the interference of other components in the blood sample.

[0023] (3) High scalability: The method is applicable to the detection of other cancer-related CTCs.

[0024] In summary, the present invention is based on the principle of enzyme-catalyzed cascade signal amplification, and designs a low-field nuclear magnetic resonance (LF-NMR) biosensor, which aims to achieve high-sensitivity detection of CTC. Through the synergistic effect of targeted triggered enzyme catalytic reaction, the addition of functionalized two-dimensional materials and acid-sensitive nanomaterials, the present invention realizes three-stage amplification of the signal, thereby significantly improving the sensitivity of LF-NMR detection. The sensor can accurately and quickly detect CTC in complex biological samples through the specific recognition of magnetic nanoparticles and circulating tumor cells, assisted by signal amplification strategy. The enzyme-catalyzed cascade reaction strategy of the present invention not only improves the detection sensitivity, but also can distinguish different cell types, eliminate the influence of background interference, and thus provide a highly accurate detection result. Therefore, the method has high sensitivity, high precision, and simple operation process, and can provide a fast and reliable detection tool for early diagnosis of clinical cancer, and has a wide range of application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the principle of the low-field nuclear magnetic resonance sensor of the present invention for detecting circulating tumor cells;

[0026] Figure 2 T cells corresponding to different concentrations of circulating tumor cells 2 Signal response diagram;

[0027] Figure 3 is the detection sensitivity curve of the low-field nuclear magnetic resonance sensor;

[0028] Figure 4 for the specificity of low-field NMR sensors;

[0029] Figure 5 The stability of low-field NMR sensors. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the accompanying drawings. Specific embodiment 1

[0032] Example 1

[0033] Preparation method and application of low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification, this method is not for the purpose of diagnosis or treatment, such as Figure 1 As shown, the following steps are included:

[0034] (1) Identification unit Fe 3 O 4 @DNA 2 Preparation of Apt

[0035] Take 5mg Fe 3 O 4-NH 2 Add 5mL of 5.0wt% glutaraldehyde aqueous solution, stir at room temperature for 2h, centrifuge and wash, and redisperse in 5mL PBS. Then add 100μL of 50μmol / L DNA2 solution and incubate at 37℃ with slow shaking for 3h. After magnetic separation, add 100μL of 5wt% BSA to block nonspecific binding sites, centrifuge and wash, and redisperse in 5mL PBS. Finally, add 100μL of 100μmol / L CTC aptamer (Apt) solution, incubate at 37℃ for 2h, and obtain Fe after magnetic separation and washing. 3 O 4 @DNA2 / Apt dispersion.

[0036] (2) Preparation of initiator GDA@GOX&DNA1

[0037] 5 mL of GDA dispersion was mixed with 5 mL of 1 mg / mL glucose oxidase (GOX) solution and 100 μL of 50 μmol / L DNA1 solution and incubated at 4 °C for 10 h to allow GOX and DNA1 to bind to the GDA surface through the reaction of amino groups with catechol groups. Subsequently, 100 μL of 5 wt% BSA was added to block nonspecific binding sites, and after centrifugation and washing, it was redispersed in 5 mL of pH = 7.4 PBS solution to obtain GDA@GOX&DNA1 dispersion.

[0038] (3) Signal unit CaO 2 @MnO 2 Preparation

[0039] 2 mg CaO 2 Disperse in 2 mL of anhydrous ethanol, add 100 μL of 10 mmol / mL MES solution, and sonicate for 30 min. After centrifugation and washing, redisperse in 2 mL of aqueous solution, add 200 μL of 3 mg / mL KMnO 4 The solution was then ultrasonicated for 30 min. After centrifugation and washing, it was redispersed in 2 mL of PBS solution to obtain CaO 2 @MnO 2 Dispersion.

[0040] (4) Preparation of LF-NMR biosensor

[0041] 50 μL Fe 3 O 4@DNA2 / Apt dispersion and 50μL GDA@GOX&DNA1 dispersion were added to 1mL of CTC-containing sample solution and incubated at 37°C for 30min. After magnetic separation and washing, they were dispersed in 500μL 50mmol / L pH=6.5PBS solution, and then 50μL universal nuclease solution was added and incubated at 37°C for 20min to release Fe bound to GDA@GOX. 3 O 4 Take 500 μL of the supernatant and add 50 μL of 5 mmol / L glucose solution and 50 μL of CaO 2 @MnO 2 Dispersion solution was incubated for 30 min. The supernatant was used for LF-NMR test, the T2 signal change was recorded, and the CTC concentration was calculated according to the formula.

[0042] Example 2

[0043] The same as the above-mentioned embodiment 1, the difference is:

[0044] (1) Identification unit Fe 3 O 4 Preparation of @DNA2 / Apt

[0045] Take 7.5 mg Fe 3 O 4 -NH 2 Add 7.5mL of 5.0wt% glutaraldehyde aqueous solution, stir at room temperature for 4h, centrifuge and wash, then redisperse in 6.9mL PBS. Add 200μL of 50μmol / L DNA2 solution, incubate at 37℃ for 2h, and wash by magnetic separation. Add 150μL of 5wt% BSA, redisperse in 6mL PBS, then add 150μL of 100μmol / L CTC aptamer (Apt) solution, incubate for 3h, and wash by magnetic separation to obtain Fe 3 O 4 @DNA2 / Apt dispersion.

[0046] (2) Preparation of initiator GDA@GOX&DNA1

[0047] 6 mL of GDA dispersion was mixed with 6 mL of 1 mg / mL GOX solution and 150 μL of 50 μmol / L DNA1 solution and incubated at 4°C for 12 h. 150 μL of 5 wt% BSA was added, and after centrifugation and washing, it was redispersed in 6 mL of pH = 7.4 PBS solution to obtain GDA@GOX&DNA1 dispersion.

[0048] (3) Signal unit CaO 2 @MnO 2 Preparation

[0049] 3 mg CaO 2 Disperse in 3 mL of anhydrous ethanol, add 150 μL of 10 mmol / mL MES solution, sonicate for 40 min, centrifuge and wash, then redisperse in 3 mL of aqueous solution, add 300 μL of 3 mg / mL KMnO 4 The solution was ultrasonicated for 40 min and then centrifuged and washed, and then redispersed in 3 mL PBS to obtain CaO 2 @MnO 2 Dispersion.

[0050] (4) Preparation of LF-NMR biosensor

[0051] 75 μL Fe 3 O 4 @DNA2 / Apt dispersion and 75μL GDA@GOX&DNA1 dispersion were added to 1.5mL CTC-containing sample solution and incubated at 37℃ for 40min. After magnetic separation and washing, they were dispersed in 750μL 50mmol / L pH=6.5PBS solution, and then 75μL universal nuclease solution was added. After incubation at 37℃ for 30min, Fe 3 O 4 Take 750 μL of the supernatant and add 75 μL of glucose solution and 75 μL of CaO 2 @MnO 2 The dispersion was incubated for 40 min and the supernatant was used for LF-NMR analysis.

[0052] Example 3

[0053] The same as the above-mentioned embodiment 1, the difference is:

[0054] (1) Identification unit Fe 3 O 4 Preparation of @DNA2 / Apt

[0055] Take 3mg Fe 3 O 4 -NH 2 Add 3mL of 5.0wt% glutaraldehyde aqueous solution, stir at room temperature for 1.5h, centrifuge and wash, then redisperse in 3.5mL PBS. Add 80μL of 50μmol / L DNA2 solution, incubate at 37℃ for 2.5h, and wash by magnetic separation. Add 70μL of 5wt% BSA, redisperse in 4.0mL PBS, then add 70μL of 100μmol / L CTC aptamer (Apt) solution, incubate for 2h, and wash by magnetic separation to obtain Fe 3 O 4 @DNA2 / Apt dispersion.

[0056] (2) Preparation of initiator GDA@GOX&DNA1

[0057] 5 mL of GDA dispersion was mixed with 5 mL of 1 mg / mL GOX solution and 100 μL of 50 μmol / L DNA1 solution and incubated at 4 °C for 10 h to allow GOX and DNA1 to bind to the GDA surface through the reaction of amino groups with catechol groups. 100 μL of 5 wt% BSA was added to block nonspecific binding sites, and after centrifugation and washing, it was redispersed in 5 mL of pH = 7.4 PBS solution to obtain GDA@GOX&DNA1 dispersion.

[0058] (3) Signal unit CaO 2 @MnO 2 Preparation

[0059] 1.5 mg CaO 2 Disperse in 1.5 mL of anhydrous ethanol, add 100 μL of 10 mmol / mL MES solution, sonicate for 25 min, centrifuge and wash, then redisperse in 1.5 mL of aqueous solution, add 150 μL of 3 mg / mL KMnO 4 The solution was ultrasonicated for 25 min and then centrifuged and washed, and redispersed in 1.5 mL PBS to obtain CaO 2 @MnO 2 Dispersion.

[0060] (4) Preparation of LF-NMR biosensor

[0061] 50 μL Fe 3 O 4 @DNA2 / Apt dispersion and 50μL GDA@GOX&DNA1 dispersion were added to 1mL of CTC-containing sample solution and incubated at 37℃ for 35min. After magnetic separation and washing, they were dispersed in 500μL 50mmol / L pH=6.5PBS solution, and then 50μL universal nuclease solution was added. After incubation at 37℃ for 25min, Fe 3 O 4 Take 500 μL of the supernatant and add 50 μL of glucose solution and 50 μL of CaO 2 @MnO 2 The dispersion was incubated for 35 min and the supernatant was used for LF-NMR analysis. Specific embodiment 2

[0063] The low-field nuclear magnetic resonance sensor prepared in the above specific embodiment 1 was placed in a low-field nuclear magnetic resonance instrument for detection, and the corresponding low-field nuclear magnetic resonance signal T under different concentrations of A549 was measured. 2 The strength of T 2The signal value is used to obtain the concentration of A549 in the test solution. The detection principle is as follows Figure 1 shown.

[0064] like Figure 2 As shown, T 2 The signal intensity decreased with increasing A549 concentration.

[0065] like Figure 3 As shown, in 10~1.0×10 6 cells / mL range, T 2 Change in signal intensity ΔT 2 (y, ms) and A549 concentration (x, cells / mL) logarithm showed a good linear relationship. The linear regression equation was y = 192*lg x + 26.99, and the square correlation coefficient R 2 =0.994. The noise is about 59 ms, and based on the signal-to-noise ratio S / N=3, the detection limit (LOD) is calculated to be 6 cells / mL. Specific embodiment three

[0067] 1.0×10 4 The experiment was carried out with HT1376, HepG2 and A549 standard solutions, blank samples and mixed samples at 100 cells / mL. Figure 4 As shown, compared with A549, the ΔT produced by other cells and blank samples 2 The mixed sample was similar to 1.0×10 4 cells / mL ΔT generated by A549 standard solution 2 The signals were roughly equal, indicating that the sensing platform has good selectivity in detecting targets in complex samples. Specific embodiment 4

[0069] The prepared nanomaterials were stored at 4 °C and the 1×10 4 cells / mL A549 produced ΔT 2 Signals are used to study the long-term stability of materials. Figure 5 As shown, after 15 days, 86.4% of the initial response value was still retained, indicating that the prepared sensor has excellent stability. Specific embodiment five

[0071] In order to evaluate the reliability of the biosensor for real sample analysis, the designed biosensor was used to detect real human 10% whole blood by the spike recovery method. The results are shown in Table 1. The spike recovery of human 10% whole blood samples was 98.9% to 103.3%, and the RSD was 1.8% to 2.4%, indicating that the sensor has excellent reliability. The results show that the sensor has good accuracy and precision and can be used to reliably detect real samples.

[0072] Table 1. Detection of A549 cells in human 10% whole blood spiked samples ( n=5)

[0073]

[0074] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Preparation and application of low-field nuclear magnetic resonance sensors for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification, which are not intended for diagnosis or treatment, and are characterized by The following steps are involved: (1) Preparation of recognition unit Fe3O4@DNA2 / Apt Take 2.5-7.5mg Fe3O4-NH2 and add it to 2.5-7.5mL 5.0wt% glutaraldehyde aqueous solution, stir at room temperature for 1-4h and wash with water, then redisperse it in 2.9-6.9mL PBS, add 50-200μL 50μmol / L DNA2 solution, incubate it at 37℃ with slow shaking for 2-4h, and then separate and wash it with magnetic force. Add 50-150μL 5wt% BSA to block nonspecific sites, redisperse it in 3.5-6.0mL PBS, add 50-150μL 100μmol / L CTC aptamer (Apt) solution, incubate it for 1-3h, wash it with magnetic force, and redisperse it in 3.5-6.0mL PBS to obtain Fe3O4@DNA2 / Apt dispersion. (2) Preparation of initiator GDA@GOX&DNA1 3.5-6.0 mL 1.0 mg / mL glucose oxidase (GOX) solution and 0.05-0.15 mL 50 μmol / L DNA1 solution were added to 4.0-6.0 mL GDA dispersion, and incubated at 4°C for 8-12 h. GOX and DNA1 were assembled on the GDA surface through the reaction of amino groups with catechol groups. 50-150 μL 5 wt% BSA was added to block non-specific sites, washed by centrifugation, and dispersed in 3.5-6.0 mL pH = 7.4 PBS solution to obtain GDA@GOX&DNA1 dispersion. (3) Preparation of signal unit CaO2@MnO2 1.0-3.0 mg CaO2 was dispersed in 1.0-3.0 mL anhydrous ethanol under ultrasound, 50-150 μL 10 mmol / mL morpholineethanesulfonic acid (MES) was added, and ultrasound was continued for 20-40 min. Subsequently, centrifugation, washing, and redispersion in 1.0-3.0 mL aqueous solution were performed, 100-300 μL 3 mg / mL KMnO4 solution was added, and ultrasound was performed for 20-40 min. Finally, centrifugation, washing, and redispersion in 1.0-3.0 mL PBS solution were performed to obtain CaO2@MnO2 dispersion. (4) Preparation of LF-NMR biosensor 25-75 μL of Fe3O4@DNA2 / Apt dispersion and 25-75 μL of GDA@GOX&DNA1 dispersion were added to 0.5-1.5 mL of sample solution containing CTC and incubated at 37°C for 20-40 min. After magnetic separation, washing and dispersion in 250-750 μL of 50 mmol / L pH=6.5 PBS solution, 25-75 μL of universal nuclease solution (25 U / μL) were added and incubated at 37°C for 15-30 min to release Fe3O4 bound to GDA@GOX. After magnetic separation, 250-750 μL of supernatant was added to 25-75 μL of 5 mmol / L glucose solution and 25-75 μL of CaO2@MnO2 dispersion and incubated for 20-40 min. After filtration, 250-750 μL of supernatant was used for LF-NMR test.

2. The preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification according to claim 1, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The synthesis method of GDA described in step (2) is as follows: 8-12 mg GO is dispersed in 20-30 mL pH = 8.5 0.01 mol / L Tris-HCl solution, and after ultrasonication for 20-40 min, 4-6 mL 1 mg / mL dopamine hydrochloride is added, and stirred at room temperature for 10-14 h to obtain GO (GDA) with polydopamine assembled on the surface. After centrifugal washing and redispersion in 8-12 mL 10 mmol / L pH = 7.4 PBS solution, a GDA dispersion is obtained.

3. The preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification according to claim 1, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The synthesis method of CaO2 described in step (3) is as follows: add 2-4 mL 1 mol / L ammonia water and 20-30 mL polyethylene glycol 200 to 4-8 mL 0.1 g / mL CaCl2 solution, then add 4-6 mL 30% H2O2 dropwise at 0.1-0.3 mL / min, and continue stirring for 1-3 h. Add 0.1 mol / L NaOH solution to adjust the pH to 10.5-12.5, wash by centrifugation, and dry at 80°C to obtain CaO2.

4. A method for preparing and using a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification according to claims 1 to 3, wherein the method is not intended for diagnosis or treatment, and is characterized in that The following steps are involved: The T2 intensity corresponding to different CTC concentrations was measured at 35°C, and the concentration of CTC in the test solution was obtained based on the T2 signal change value (ΔT2).

5. The preparation method and application of a low-field nuclear magnetic resonance sensor for detecting circulating tumor cells based on enzyme-catalyzed cascade signal amplification according to claim 4, wherein the method is not for the purpose of diagnosis or treatment, and is characterized in that The T2 signal change value (ΔT2) is calculated according to the following formula: ΔT2=T2(negative)-T2(positive), where T2(negative) is the average T2 when there is no CTC in the sample, and T2(positive) is the average T2 when there is CTC in the sample.

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