CN116173250A

CN116173250A - Targeting molecular probe based on aptamer and preparation method and application thereof

Info

Publication number: CN116173250A
Application number: CN202211016917.1A
Authority: CN
Inventors: 谭蔚泓; 丁玎; 肖泽宇; 刘建军
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-08-24
Filing date: 2022-08-24
Publication date: 2023-05-30

Abstract

The invention discloses a targeting molecular probe based on a nucleic acid aptamer, a preparation method and application thereof, and relates to the field of biotechnology and molecular imaging. The preparation method comprises the following steps: preparing a nucleic acid aptamer-nuclide molecular probe precursor and labeling with a short half-life nuclide molecular probe. Molecular probes [ ⁶⁸ Ga]NOTA-SGC8 comprising SGC8 aptamer sequence, NOTA as ligand coupler and ⁶⁸ ga radiocontrast agent. The application of the molecular probe in molecular imaging. The invention adopts aptamer targeting molecules to replace traditional antibody targeting molecules. The aptamer sequence can be synthesized entirely using a DNA synthesizer in the solid phase; the sequence can be personalized; the batch-to-batch variation is small. The reaction condition is simple, the stability is good, and the reaction is not influenced by temperature and pH value. The probe preparation method can be universally used for creating molecular probes aiming at other molecular targets and different nuclides.

Description

Targeting molecular probe based on aptamer and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology and molecular imaging, in particular to a targeting molecular probe based on a nucleic acid aptamer, and a preparation method and application thereof.

Background

Molecular imaging, i.e., the use of molecular imaging contrast agents and tools, visualizes, characterizes and measures biological processes at the molecular and cellular level of the human body or other living system. Positron emission tomography (Positron emission tomography, PET) has greatly improved the global medical health level since its entry into clinical use as the most representative molecular imaging tool. In recent years, with the gradual discovery of the pathogenesis of cancer molecules and further research into the host immune system, molecular targeted therapies (e.g., small molecule inhibitors and monoclonal antibodies, mAbs) and immunotherapies (e.g., immune checkpoint inhibitors) have been developed. The clinical application of these new therapies is changing the therapeutic prospects of many cancers. In the age of molecular targeted therapy and cancer immunotherapy, PET imaging based on conventional radiotracers is difficult to meet the requirements of accurate medicine. For example, 18F-fluorodeoxyglucose (18F-FDG) PET/Computed Tomography (CT) has been integrated into several important criteria for predicting and assessing the response of targeted therapies or immunotherapies. However, studies have reported that parameters acquired by PET/CT based on 18F-FDG contrast agents, such as SUVmax and SUVmean, are not highly correlated with clinical response profiles of immunotherapeutic regimens. Furthermore, the use of PET imaging based on such contrast agents to distinguish between adverse events associated with immunity (e.g., sarcoidosis) and false positives is very challenging and can easily lead to false positives in the condition.

The antibody-based molecular imaging probes help to observe the expression and pharmacokinetics of the corresponding targets of therapeutic monoclonal antibodies in vivo. Antibody-mediated contrast PET imaging has significant advantages in terms of image quality, spatial resolution, and quantification over several antibody contrast agents currently in clinical existence for Single Photon Emission Computed Tomography (SPECT) imaging. In recent years, with the development of science and technology, researchers have developed nuclide molecular probes based on targeting molecules such as small molecules and nucleic acid aptamers. Nucleic acid aptamers have a number of advantages over antibodies, for example: 1) The aptamer is a nucleic acid sequence, can be chemically synthesized, and has low cost and small batch-to-batch difference; 2) The aptamer has good chemical and thermal stability, can be functionalized in various coupling modes, and is beneficial to probe development; 3) The nucleic acid aptamer has high metabolism speed, and can quickly clear out the body and reduce radiation injury by matching with a short half-life nuclide label. The nucleic acid aptamer-based probe can be specifically combined with a tumor marker, and can realize molecular imaging of the tumor tissue high-expression biomarker through PET/CT imaging, so as to provide molecular diagnosis information of the tumor tissue. The imaging molecular diagnosis can provide complete tumor molecular physiological information, and the problems that diagnosis information is not complete and the like possibly exist in pathological section immunohistochemistry are avoided. In addition, the molecular image diagnosis based on the PET/CT technology can carry out imaging and molecular typing on the primary tumor focus and the metastasis in an early stage in a noninvasive manner, and provides a rich and reliable diagnosis basis for the implementation of corresponding treatment schemes.

Malignant bladder tumor is one of male high-incidence malignant tumor, which threatens human life and health for a long time, and the accurate medical treatment means based on the emerging therapies such as immunotherapy at present has been well represented in bladder tumor. However, the formulation of reasonable treatment schemes and targeted drug selection requires comprehensive marker expression condition analysis of tumors, and the development of a malignant bladder tumor molecular imaging probe based on a nucleic acid aptamer is expected to provide a new and efficient diagnostic tool for the field.

Traditional Chinese medicine ¹⁸ F-FDG contrast agent can not perform targeted imaging on specific targets in tumors, can not provide comprehensive molecular diagnosis information for accurate medicine, and is difficult to meet tumor physiological information required by targeted therapy. The targeting probe based on the antibody also has the problems of high preparation difficulty, long half-life period, high radiation risk and the like.

Accordingly, those skilled in the art have been working to develop a nucleic acid aptamer-based nuclide molecular probe, and a preparation method and application thereof.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to develop a nuclide molecular probe based on aptamer, and a preparation method and application thereof.

In order to achieve the above purpose, the invention provides a preparation method of a PTK7 receptor protein targeting aptamer molecular probe, which comprises the following steps:

step 1, preparing a nucleic acid aptamer-nuclide molecular probe precursor;

and 2, labeling the molecular probe precursor obtained in the step 1 by using a short half-life nuclide molecular probe.

Further, step 1 further includes:

step 1.1, solid phase synthesis of aptamer containing target nucleic acid for PTK7 receptor protein by using a DNA synthesizer;

step 1.2, coupling the nucleic acid aptamer obtained in the step 1.1 with a nuclide chelating ligand.

Further, the aptamer-nuclide molecular probe precursor is aptamer SGC8.

Further, the aptamer SGC8 sequence is: 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3'.

Further, the chelating ligand in step 1.2 includes Deferoxamine (Deferoxamine), 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA).

Further, step 2 further includes:

step 2.1, molecular Probe precursor, radioactivity ⁶⁸ Mixing Ga hydrochloric acid solution and a reaction solvent;

step 2.2, obtained by reaction ⁶⁸ Ga-labeled aptamer-nuclide molecular probes.

Further, step 2.1 further comprises:

(1) Leaching germanium gallium generator with hydrochloric acid to obtain radioactivity ⁶⁸ Ga hydrochloric acid solution a;

(2) Dissolving the aptamer in a sodium acetate buffer solution, and adjusting the pH to obtain a solution b.

Further, step 2.2 further comprises:

(3) Mixing the solution b with the solution a, and carrying out oscillation reaction for 1 hour at 90 ℃ to obtain a product c;

(4) And (3) purifying the product c obtained in the step (3) to obtain the radionuclide molecular probe.

Further, in the solution a of the step (1) ⁶⁸ Ga hydrochloric acid solution is added in an amount of 2mC of radioactive dosei-8 mCi, e.g., 2mCi, 4mCi, 6mCi, or 8mCi.

Further, the germanium gallium generator in the step (1) is leached ⁶⁸ The volume of the Ga hydrochloric acid solution is 1-2 mL;

further, the germanium gallium generator in the step (1) is leached ⁶⁸ The volume of the Ga hydrochloric acid solution is 1mL, 1.5mL, or 2mL.

Further, the volume of the buffer solution for dissolving the SGC8 aptamer in the step (2) is 0.02-0.1 mL;

further, the buffer solution in which the SGC8 aptamer is dissolved in step (2) has a volume of 0.02mL, 0.05mL, or 0.1mL.

Further, the mixing volume ratio of the solution a to the solution b in the step (3) is 10: 1-20: 1, a step of;

further, the mixing volume ratio of the solution a to the solution b in the step (3) is 1mL:0.1mL or 2mL:0.1mL.

Further, the aptamer SGC8 precursor in step (2) is a lyophilized powder with the chelating ligand NOTA.

Further, the reaction time of the step (3) is 1 to 2 hours;

further, the reaction time of step (3) is 1 hour, 1.5 hours or 2 hours.

Further, product c in step (4) was purified using a GE healthcare Nap desalting purification column, methods of use being referred to desalting column product instructions.

The invention also provides a PTK7 receptor protein targeting aptamer molecular probe prepared by the preparation method of the PTK7 receptor protein targeting aptamer molecular probe, and the molecular probe is [ the ⁶⁸ Ga]NOTA-SGC8 comprising SGC8 aptamer sequence, NOTA as ligand coupler and ⁶⁸ a Ga radiocontrast agent; wherein SGC8 is a nucleic acid aptamer sequence which is specifically combined with a high expression PTK7 receptor protein in bladder tumor, ⁶⁸ ga-radiocontrast agents are used for positron emission computed tomography (PET).

The invention also provides application of the PTK7 receptor protein targeting aptamer molecular probe prepared by the preparation method of the PTK7 receptor protein targeting aptamer molecular probe in molecular imaging.

Further, the labeling species may be any of Tc-99m, ga-68, F-18, I-123, I-125, I-131, I-124, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, Y-86, mn-52, sc-44; or any one of Lu-177, Y-90, ac-225, as-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra-223, ru-106, na-24, sr-89, tb149, th-227, xe-133, yb-169, yb-177.

The invention also provides a nucleic acid aptamer sequence with similar targeting recognition and binding of specific molecular markers as an antibody, and the nucleic acid aptamer can be combined with the PTK7 receptor protein overexpressed on the surface of malignant bladder tumor.

The aptamer provided by the invention can be used for PET/CT molecular imaging. By performing nuclide chelate ligand coupling and further radionuclide labeling on SGC8 aptamer, a PET molecular probe aiming at a specific molecular marker PTK7 can be constructed. Finally, the tumor is successfully lightened in the malignant bladder cancer patient, and the diagnosis efficacy of the probe is verified.

In preferred embodiment 1 of the present invention, the steps for preparing a nucleic acid aptamer molecular probe targeting malignant bladder tumor are described in detail;

in another preferred embodiment 2 of the present invention, details are described ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶ 8Ga]Stability of NOTA-SGC8 molecular imaging probes;

in another preferred embodiment 3 of the present invention, details are described ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]Cell specific binding experiments of NOTA-SGC8 molecular image probes;

in another preferred embodiment 4 of the present invention, details are described ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ₆ 8Ga]Tumor specific imaging of NOTA-SGC8 molecular imaging probes in bladder cancer patients.

The invention has the following beneficial effects:

the invention realizes noninvasive visual detection of PTK7 receptor protein expression by creating the PTK7 specific novel SGC8 aptamer PET imaging probe [68Ga ] NOTA-SGC8, further realizes noninvasive diagnosis of malignant bladder tumor, and has the advantages of simple preparation process, low cost, high specificity, high stability, high imaging signal-to-time ratio, easy clinical transformation and the like.

The invention adopts aptamer targeting molecules to replace traditional antibody targeting molecules. The aptamer sequence can be synthesized entirely using a DNA synthesizer in the solid phase; the sequence can be personalized; the batch-to-batch variation is small. Compared with the traditional antibody probe preparation, the reaction condition is simple; the probe synthesis precursor and the probe have good stability, and are not influenced by temperature and pH value. Nuclide-labeled PET imaging probe based on SGC8 aptamer [ ⁶⁸ Ga]NOTA-SGC8 can be used for noninvasive visual molecular diagnosis of malignant bladder tumors. The probe preparation method disclosed by the invention can be universally used for creating molecular probes for other molecular targets and different nuclides, and has wide application prospect and very high clinical conversion value in future diagnosis and accurate treatment of tumor tissues.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a flow chart showing a method for preparing SGC8 nucleic acid aptamer according to a preferred embodiment 1 of the present invention;

FIG. 2 is a schematic representation of the coupling of a nucleic acid molecule of a preferred embodiment 1 of the invention to a NOTA ligand;

FIG. 3 is a high performance liquid chromatogram of an SGC8 aptamer of preferred embodiment 3 of the present invention after NOTA ligand coupling;

FIG. 4 is a high resolution mass spectrum of SGC8 nucleic acid aptamer of preferred embodiment 3 of the present invention after NOTA ligand coupling;

FIG. 5 is a preferred embodiment 3 of the present invention ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]A NOTA-SGC8 probe stability investigation chart;

FIG. 6 is a comparative example of the present inventionExample 4 ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]NOTA-SGC8 probe cell specific binding patterns;

FIG. 7 is a preferred embodiment 5 of the present invention ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]A molecular image map of PET/CT imaging of the NOTA-SGC8 molecular probe 30 minutes after bladder perfusion of the bladder cancer patient;

FIG. 8 is a PET/CT imaging molecular image of a preferred embodiment 5 using a 18F labeled 18F-FDG control probe in the clinic;

FIG. 9 shows the result of a pathological section of bladder cancer patient in accordance with the preferred embodiment 5.

Detailed Description

The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.

The aptamer referred to in the application is a segment of artificially synthesized single-stranded DNA or RNA, which is usually obtained by in vitro screening by an exponential-rich ligand systematic evolution technique (SELEX) method, and has higher affinity and specificity with a target ligand.

The invention discloses a novel molecular image probe for malignant bladder tumor ⁶⁸ Ga]NOTA-SGC8, wherein SGC8 is a specific binding aptamer sequence of a high-expression PTK7 receptor protein in bladder tumor, NOTA is a ligand coupling agent, ⁶⁸ ga (T1/2=68 min) is a radiocontrast agent for positron emission computed tomography (PET).

Example 1 preparation of aptamer molecular probes targeting malignant bladder tumor

The preparation of the aptamer molecular probe for targeting malignant bladder tumor specifically comprises the following steps:

(1) As shown in fig. 1, adenine, guanine, cytosine and thymine are synthesized into bladder tumor specific SGC8 aptamer by a solid phase synthesis method of a DNA synthesizer, and simultaneously an amino group is introduced into the 5' end of the aptamer for further coupling, while a specific sequence of a control Library sequence (Library) is shown as follows, wherein N is a random base;

SGC8：5’-(NH2)-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3’

Library：5’-(NH2)-ATC TAA CTG ANN NNN NNN NNN NNN NNN NNN NNN CGG TTA GA-3’

(2) Preparation of 0.1M sodium phosphate and 0.1M sodium bicarbonate Mixed solution of 3.8g Na ₃ PO ₄ +0.84g NaHCO ₃ The volume was set to 100mL with ultrapure water. In addition, small molecule CNS-NOTA 46.1mg was weighed to prepare 0.7mL DMF solution, 94.9mg of nucleic acid powder was weighed and dissolved to 9mL with the prepared sodium phosphate/sodium bicarbonate solution. As shown in fig. 2, mixing the NOTA solution and the nucleic acid solution to a constant temperature shaking table for oscillation, reacting for 1 hour at 20-25 ℃, stopping the reaction, purifying by using HPLC to obtain NOTA-SGC8 aptamer powder, verifying the molecular weight of the product and performing HPLC analysis, and freeze-drying for storage;

(3) 100. Mu.g of NOTA-SGC8 aptamer powder is dissolved in 100. Mu.l of 0.25M sodium acetate buffer solution;

(4) Eluting germanium gallium generator with 4ml hydrochloric acid solution with concentration of 0.05M, collecting the interruption 2ml active about 5mCi ⁶⁸ Ga leaches;

(5) In step 4 ⁶⁸ Mixing Ga leacheate with the aptamer precursor solution in the step 3, vibrating and mixing for 1 hour at room temperature, then purifying and separating the free product by using PBS as a mobile phase and using a pre-balanced PD-10 desalting column (GE Healthcare) ⁶⁸ Ga ion, finally using a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) the radiochemical purity of the probe was determined (Radiochemical purity, RCP).

Example 2 investigation ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶ 8Ga]NOTA-SGC8 molecular image probe stability

⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶ 8Ga]Stability investigation of NOTA-SGC8 molecular image probe. The method specifically comprises the following steps:

(1) Fetch [ ⁶⁸ Ga]100 μCi of NOTA-SGC8 aptamer molecular probe is respectively dissolved in 1ml of packaged 1XPBS bufferThe solution was incubated with fetal bovine serum FBS and with 37 ℃ for 1 hour;

(2) Experimental Balb/c white mice were injected via the tail vein with about 100. Mu. Ci [ [ ⁶⁸ Ga]Urine is collected 1 hour after the NOTA-SGC8 aptamer molecular probe;

(3) After incubation, the probe in the step 1 and urine collected from the mice in the step 2 are analyzed by HPLC, and the stability of the molecular probe is inspected by inspecting the characteristic peak intensity of specific retention time. The high performance liquid chromatogram after the SGC8 aptamer is subjected to NOTA ligand coupling is shown in figure 3, and the retention time is 8.444 minutes; the high resolution mass spectrum of SGC8 aptamer after NOTA ligand coupling is shown in figure 4, ⁶⁸ ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]The mass number of the NOTA-SGC8 molecular image probe is 13261.6; ⁶⁸ ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]Stability study of the NOTA-SGC8 probe as shown in FIG. 5, the probe remained intact after incubation in PBS, FBS for 1 hour, while urine was collected one hour after intravenous injection into the tail of the mouse, and the complete probe structure was still detectable.

Example 3 cell specific binding assay

⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]The cell specific binding experiment of the NOTA-SGC8 molecular image probe specifically comprises the following steps:

(1) Pre-subculturing by using a bladder cancer tumor cell line positive to PTK7 through Western blot;

(2) Tumor cells were harvested and split equally to 106 cells per tube. Will respectively [ ⁶⁸ Ga]NOTA-SGC8 molecular imaging probe and contrast thereof ⁶⁸ Ga]Adding the NOTA-Library solution to the cell suspension;

(3) After the cells were incubated at 4℃for 4 hours, the supernatant was washed off by centrifugation and the cells were resuspended in 0.5ml of buffer solution;

(4) Radioactivity of the samples was measured using a gamma counter (2470Wizard2,Perkin Elmer,Massachusetts,USA). The ability of molecular probes to specifically bind to cells was examined by comparing the direct radioactive doses of the samples. ⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]The cell-specific binding results of the NOTA-SGC8 probe are shown in FIG. 6, where are represented bySignificant differences, results indicate that compared to control probes [ [ ⁶⁸ Ga]The NOTA-SGC8 probe is capable of specifically binding to target cells.

EXAMPLE 4 specific imaging

⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]Tumor specific imaging of NOTA-SGC8 molecular imaging probes in bladder cancer patients. The method specifically comprises the following steps:

(1) The clinical experiment group patients firstly carry out physical examination including vital signs, routine hematuria, liver and kidney functions, blood HCG (female) and electrocardiogram, which are all completed within one week before PET/CT examination;

(2) PET/CT static acquisition images using a phantom (United Imaging uMI 780), the patient default perfusion dose was 3mCi. Double checking the patient's name, sex, age, PET number and weight before administering the imaging probe to the patient;

(3)[ ⁶⁸ Ga]the NOTA-SGC8 molecular imaging probe was administered by transurethral infusion at the bedside and instructed to empty the bladder as much as possible about 3 minutes prior to infusion;

(4) Static imaging begins with CT scans to attenuate corrections for later PET scans, ranging from overhead to knees. The acquisition time for transverse PET scan was 2 minutes/slice. The actual perfusion dose and time were recorded after the PET image acquisition. CT scan and reconstruction parameters: 120kV whole-body automatic mA. The layer thickness was 3.0mm, the FOV was 500mm, and the abdomen was reconstructed as B_SOFT_B. PET scan and reconstruction parameters: 2 minutes per whole body acquisition, reconstruction parameters OSEM, iteration number 3, subset 20, image size 192×192, smoothness Smooth3 and Gaussian half-height width 3.0;

(5) The next day after the imaging is finished, physical examination is carried out, including vital signs, routine hematuria, liver and kidney functions and electrocardiogram;

(6) The same patient was subjected to standardized 18F-FDG intravenous PET/CT molecular imaging one week after the first perfusion imaging.

⁶⁸ Ga-labeled SGC8 nucleic acid aptamer [ ⁶⁸ Ga]The molecular image of the NOTA-SGC8 molecular probe for PET/CT imaging 30 minutes after bladder perfusion in a bladder cancer patient is shown in FIG. 7,white arrows indicate tumor sites.

A molecular image of PET/CT imaging using an 18F-labeled 18F-FDG control probe for intravenous administration is shown in FIG. 8, with white arrows indicating tumor sites. [ ⁶⁸ Ga]The NOTA-SGC8 probe can specifically lighten the tumor of a patient, and the related result is clinically used ¹⁸ F-FDG contrast agent was validated.

FIG. 9 is a result of a pathological section of the bladder cancer patient in FIG. 7 after surgery, confirming that the patient is overexpressed [ ⁶⁸ Ga]NOTA-SGC 8min probe target PTK7 receptor protein.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims

1. A method for preparing a PTK7 receptor protein targeting aptamer molecular probe, which is characterized by comprising the following steps:

step 1, preparing a nucleic acid aptamer-nuclide molecular probe precursor;

2. The method of claim 1, wherein step 1 further comprises:

step 1.1, solid phase synthesis of a nucleic acid aptamer comprising a target for the PTK7 receptor protein using a DNA synthesizer;

3. The method of claim 2, wherein the sequence of the aptamer is: 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3'.

4. The method of claim 2, wherein the chelating ligand in step 1.2 comprises Deferoxamine (Deferoxamine), 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA).

5. The method of claim 1, wherein step 2 further comprises:

step 2.1, the molecular probe precursor and radioactivity ⁶⁸ Mixing Ga hydrochloric acid solution and a reaction solvent to obtain a mixed solution;

step 2.2, the mixed solution obtained in the step 2.1 is obtained through reaction ⁶⁸ Ga-labeled aptamer-nuclide molecular probes.

6. The method of claim 5, wherein step 2.1 further comprises:

(1) Leaching germanium gallium generator with hydrochloric acid to obtain the radioactivity ⁶⁸ Ga hydrochloric acid solution a;

(2) And dissolving the molecular probe precursor in a sodium acetate buffer solution, and regulating the pH value to obtain a solution b.

7. The method of claim 6, wherein step 2.2 further comprises:

(4) Purifying the product c obtained in the step (3) to obtain the radionuclide molecular probe.

8. A PTK7 receptor protein targeting aptamer molecular probe prepared by the method according to any one of claims 1-7, wherein the molecular probe is [ the ⁶⁸ Ga]NOTA-SGC8 comprising SGC8 aptamer sequence, NOTA as ligand coupler and ⁶⁸ a Ga radiocontrast agent; wherein the SGC8 isHigh expression of the nucleic acid aptamer sequence specifically bound to the PTK7 receptor protein in bladder tumors, the ⁶⁸ Ga-radiocontrast agents are used for positron emission computed tomography (PET).

9. The use of the molecular probe according to claim 8 in molecular imaging.

10. The use according to claim 9, wherein the labeling species of the molecular probe is any one of Tc-99m, ga-68, F-18, I-123, I-125, I-131, I-124, in-111, ga-67, cu-64, zr-89, C-11, lu-177, re-188, Y-86, mn-52, sc-44; or any one of Lu-177, Y-90, ac-225, as-211, bi-212, bi-213, cs-137, cr-51, co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, I-131, ir-192, fe-59, pb212, mo-99, pd-103, P-32, K-42, re-186, re-188, sm-153, ra-223, ru-106, na-24, sr-89, tb149, th-227, xe-133, yb-169, yb-177.