A sort of 19 F-PDA-DNA-DTPA / Gd nanosensing probe, its preparation method, and its application in fluorine magnetic resonance imaging.

Abstract

This invention provides 19 F-PDA-DNA-DTPA / Gd nanosensing probe, its preparation method, and its application in fluorine magnetic resonance imaging: This nanosensing probe consists of an MR signal source, a quencher, and a response linker. The MR signal source is a high-density fluorine perfluorocarbon nanoparticle, the quencher is Gd-DTPA, and the response linker is a DNA aptamer composed of a single-stranded DNA S1 and its complementary strand S2, with nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. As a responsive nanoparticle platform, it is only visible upon activation by a specific biological trigger. Its biological application is demonstrated by targeting thrombin with the aptamer and exhibiting a strong and selective response to thrombin in live mice.

Description

Technical Field

[0001] This invention belongs to the field of biological probe technology, specifically relating to a... 19 F-PDA-DNA-DTPA / Gd nanosensing probe, its preparation method, and its application in fluorine magnetic resonance imaging. Background Technology

[0002] Magnetic resonance imaging (MRI) is a non-invasive diagnostic technique used to visualize anatomical structures and physiological functions. The development of MRI molecular imaging probes is crucial for the diagnosis and prognostic assessment of various adverse health conditions. Among these, gadolinium (Gd)-based contrast agents are the most extensively studied. Some contrast agents enhance MRI by altering the relaxation rate of protons in surrounding water molecules. 1 The contrast of H MRI. Although enhanced contrast 1 H MRI has great application value in molecular imaging, but its low sensitivity and large background signal limit its application.

[0003] Fluorine magnetic resonance imaging (FMRI) 19 fMRI is 1 An emerging alternative to 1H MRI, it produces a low background signal due to its extremely low endogenous fluoride concentration. This enables the direct and quantitative detection of fluoride-containing... 19 F probe. 19 FMR images can be compared with anatomical structures 1 Overlaying HMR images provides complementary information. Fluorine-containing nanoparticles serve as... 19 Imaging agents for fMRI have attracted considerable attention, as they can encapsulate high concentrations of fluorinated molecules in an aqueous medium. The encapsulating materials used include phospholipids, polymeric surfactants, or solid coatings (such as silica). Phospholipid-coated perfluorocarbon nanoemulsions have been used for in vitro cell labeling and tracking of macrophage uptake in vivo. Targeted nanoemulsions have been used to image biomarkers including integrin ανβ3, fibrin clots, and α2-antiplasticase.

[0004] In the field of molecular imaging, "on" sensors are of particular interest, where the signal of the imaging probe is quenched before interacting with a specific analyte and then turned on after the interaction. 19 F-signal. These probes are more analyte-specific than targeted probes because targeted probes generate a signal regardless of whether they bind to the target. Kikuchi, in his pioneering work, reported several silica-coated perfluorocarbon core-shell nanoparticle "on" probes, including gadolinium ion (Gd) 3+The complex is coupled to the surface of nanoparticles. These probes achieve signal on / off via a distance-dependent paramagnetic relaxation enhancement effect; the "on" signal is obtained when the complex cleaves from the surface. This mechanism has been used to detect reducing environments and various hydrolases, including visualization of caspase-3 / 7 activity in live mice. However, a drawback of these probes is that a single gadolinium ion (Gd) is visible. 3+ The complexes cannot strongly quench the signal of perfluorocarbon nanoparticles, so thousands of complexes are needed to quench a single nanoparticle. This limits the sensitivity of the probe, as a large number of fragments are required to reach a detectable signal level. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a [missing information]. 19 F-PDA-DNA-DTPA / Gd nanosensing probe.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] (i) It consists of an MR signal source, a quenching agent, and a response connector;

[0010] (ii) The MR signal source is high-density fluorine perfluorocarbon nanoparticles, the quencher is Gd-DTPA, and the response linker is a DNA aptamer composed of a single DNA strand S1 and its complementary strand S2, with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] Another object of the present invention is to provide a 19 Preparation method of F-PDA-DNA-DTPA / Gd nanosensing probe.

[0012] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including,

[0013] 100 mg of cetyltrimethylammonium bromide was dispersed in 10–15 mL of water and ultrasonicated to obtain a dispersion.

[0014] Add 55–65 μL of perfluorocarbon compound to the dispersion, and then sonicate at 50 °C for 2 hours to obtain a nanoemulsion suspension.

[0015] Add 90–110 μL of NH3·H2O and 25–35 mL of water to the nanoemulsion suspension, followed by 1–3 mL of a DA aqueous solution with a concentration of 45–55 mg / mL, and stir to obtain the desired solution. 19 F-PDA solution;

[0016] 19 F-PDA and single-stranded DNA S1 were reacted in Tris buffer with vigorous stirring, centrifuged and washed. The resulting product and 8–12 mM complementary DNA S2 were hybridized in PBS buffer, followed by centrifugation and washing to obtain... 19 F-PDA-DNA;

[0017] 8–12 mmol of DTPA-DA dissolved in 19 A mixed solution was obtained by dissolving F-PDA-DNA in an aqueous solution. The pH of the mixed solution was adjusted, and DTPA-modified DNA was obtained by centrifugation. 19 F-PDA-DNA nanoprobes;

[0018] DTPA modified 19 F-PDA-DNA nanoprobes were washed and dispersed in deionized water to obtain DTPA-modified [products / products]. 19 F-PDA-DNA nanoprobe solution;

[0019] Stirring DTPA-modified [material] at room temperature 19 F-PDA-DNA nanoprobe solution, and GdCl3 was added to it for Gd... 3+ Complex, adjust pH, centrifuge and wash to remove unreacted Gd. 3+ Ultimately obtained 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0020] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probe, wherein the ultrasonic treatment time for the dispersion obtained by ultrasonic treatment is 30-60 min.

[0021] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probe, wherein: the ultrasonic treatment to obtain the nanoemulsion suspension takes 1-2 hours and the ultrasonic temperature is 45-55℃.

[0022] As described in this invention 19A preferred embodiment of the preparation method of the F-PDA-DNA-DTPA / Gd nanosensing probe, wherein: 19 The reaction time of F-PDA with single-stranded DNA S1 in Tris buffer with vigorous stirring is 10–14 h.

[0023] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probe, wherein the reaction temperature of the hybridization reaction is 35-40℃.

[0024] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probe, wherein the reaction time of the hybridization reaction is 1.5 to 2.5 h.

[0025] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probes, wherein the pH of the mixed solution is adjusted to 6.5-7.5 by using NaOH.

[0026] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probes, wherein: the DTPA-modified... 19 The stirring time for the F-PDA-DNA nanoprobe solution is 2.5–3.5 h.

[0027] As described in this invention 19 A preferred embodiment of the preparation method of F-PDA-DNA-DTPA / Gd nanosensing probes, wherein: after adjusting the pH, centrifugation and washing are performed to remove unreacted Gd. 3+ The pH should be adjusted to 6-7.

[0028] Beneficial effects of this invention:

[0029] In summary, this invention designs a 19 The F-PDA-DNA-DTPA / Gd nanoprobe, acting as a responsive nanoparticle platform, is only visible upon activation by a specific biological trigger. Its biological applications are demonstrated through aptamer-targeted thrombin targeting and a strong and selective response to thrombin in live mice. Furthermore, the selected aptamer DNA is used for the first time in photothermal desorption, eliminating the need for enzymatic response; the probe itself dissociates via photothermal action, enabling the signal to be generated from nothing.

[0030] This invention uses 19Preliminary demonstration of in vivo sensing using F-PDA-DNA-DTPA / Gd nanoprobes, combining 19 In the fields of fMRI, aptamer sensors, and molecular diagnostics, this provides a reference for developing diagnostic probes that will be activated in the presence of important disease-related analytes. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0032] Figure 1 This invention is based on aptamers 19 fMRI biosensor probe design diagram.

[0033] Figure 2 This is a heatmap and electrophoresis diagram of the base pairing probability in the DNA aptamer of this invention.

[0034] Figure 3 For the present invention 19 TEM and elemental maps of F-PDA-DNA-DTPA / Gd.

[0035] Figure 4 For the present invention 19 DLS analysis diagram of F-PDA-DNA-DTPA / Gd.

[0036] Figure 5 For the present invention 19 F-PDA-DNA-DTPA / Gd probe magnetic resonance imaging assessment.

[0037] Figure 6 The present invention provides nanoprobes of different concentrations. 19 Cell viability when F-PDA-DNA-DTPA / Gd is co-incubated with cells.

[0038] Figure 7 For the present invention 19 Blood compatibility analysis of F-PDA-DNA-DTPA / Gd at different concentrations.

[0039] Figure 8 For the present invention 19 Blood analysis data of the F-PDA-DNA-DTPA / Gd nanoprobe group and the control group.

[0040] Figure 9 Injection for the present invention 19Mouse organ and tissue sections at different time points after F-PDA-DNA-DTPA / Gd. (Scale bar: 100m)

[0041] Figure 10 Injection for the present invention 19 Mice treated with F-PDA-DNA-DTPA / Gd in different ways 1 H / 19 F magnetic resonance imaging image.

[0042] Figure 11 This invention relates to the co-incubation of thrombin with different concentrations under 480nm excitation. 19 Fluorescence spectrum of DOX released by the F-PDA-DNA-DTPA / Gd probe.

[0043] Figure 12 For the present invention 19 Laser confocal images of F-PDA-DNA-DTPA / Gd probes processed using different methods. (Scale bar: 10 μm)

[0044] Figure 13 The samples prepared under different amounts of DA aqueous solution in Comparative Example 1 of this invention 19 Transmission electron microscopy image of F-PDA-DNA-DTPA / Gd nanoprobe. Detailed Implementation

[0045] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0046] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0048] Unless otherwise specified, all raw materials used in this invention are commercially available in the art. Specifically:

[0049] Hexadecyltrimethylammonium bromide, eicosfluoro-15-crown-5-ether, dopamine hydrochloride (DA, 99% purity), tetraethyl silicate, ethyl acetate, propidium iodide (PI), doxorubicin hydrochloride (DOX, 98% purity), and calcein AM were purchased from Aladdin, while diethylenetriaminepentaacetic dianhydride (DTPA-DA, 95%) and gadolinium(III) hexahydrate (GdCl3, 99.9%) were purchased from Alfa Aesar.

[0050] All oligonucleotides used in this study were synthesized by Shanghai Sangon Biotech Co., Ltd. and purified by high-performance liquid chromatography (HPLC) (sequences are shown in Table 1).

[0051] Table 1 Oligonucleotide Sequences

[0052]

[0053] The present invention refers to the following method for 19 The relevant properties of F-PDA-DNA-DTPA / Gd were evaluated using tests:

[0054] In vitro cytotoxicity assessment

[0055] Cells were added to 96-well cell culture plates containing culture medium and incubated at 37°C for 24 hours under conditions of 5% carbon dioxide and 95% humidity to allow the cells to spread throughout the plate. The culture medium was then removed, and cells containing different concentrations of... 19 Incubate the cells in F-PDA-DNA-DTPA / Gd medium for 24 hours (note that the cell density in the 96-well plate must be greater than 70%). After washing twice with PBS, add the cells to 10% CCK-8 medium and incubate for another 2 hours. Finally, measure the absorbance at 450 nm using a microplate reader and calculate the cell viability using the following formula:

[0056] Cell viability (%) = (Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells) × 100%;

[0057] Blood test

[0058] Two groups (n=5) of healthy mice were anesthetized with isoflurane; one group was injected with... 19 The mice were treated with F-PDA-DNA-DTPA / Gd solution, while the other group received no treatment. Behavioral and weight changes in the mice were observed after injection. Blood samples were collected one month later for blood testing.

[0059] Construction of tumor models

[0060] HepG2 cells were cultured in DMEM medium at 37°C with 5% carbon dioxide and 95% humidity. When the cell number reached the desired level, the cells were repeatedly digested and washed with PBS, and then dispersed in serum-free DMEM medium. A total volume of 2 × 10⁶ cells / day was added. 6 4T1 cells were injected intramuscularly into each BALB / c mouse.

[0061] Example 1

[0062] This embodiment provides a 19 The preparation method of F-PDA-DNA-DTPA / Gd nanoprobes is as follows:

[0063] 1) 19 Synthesis of F-PDA;

[0064] 100 mg of cetyltrimethylammonium bromide (CTAB) was dispersed in 12 mL of water and sonicated for 30 minutes to obtain a dispersion.

[0065] 60 μL of perfluorocarbon compound (PFCE) was added to the dispersion, followed by ultrasonic treatment at 50 °C for 2 hours to obtain a nanoemulsion suspension;

[0066] Add 100 μL of NH3·H2O and 30 mL of water to the nanoemulsion suspension, followed by 2 mL of 50 mg / mL DA aqueous solution, and stir for 24 h to obtain... 19 F-PDA.

[0067] 2) 19 The synthesis of F-PDA-DNA;

[0068] 19 F-PDA and single-stranded DNA S1 were reacted in Tris buffer (10 mM, pH 8.5) with vigorous stirring for 12 h. The mixture was then centrifuged at 10,000 rpm and washed three times. The resulting product and 10 mM complementary DNA S2 were hybridized in PBS buffer (NaCl 136.89 mM, KCl 2.67 mM, Na2HPO4 8.1 mM, KH2PO4 1.76 mM, pH 7.4) at 37 °C for 2 h. The mixture was then centrifuged at 10,000 rpm and washed three times with deionized water to obtain the desired product. 19 F-PDA-DNA;

[0069] 3) 19 Synthesis of F-PDA-DNA-DTPA / Gd nanoprobes;

[0070] 10 mmol DTPA-DA dissolved in 19 A mixed solution was obtained by adding F-PDA-DNA to an aqueous solution;

[0071] NaOH was added to the mixture to adjust the pH to 7, and after stirring for 24 hours, the DTPA-modified product was obtained by centrifugation. 19 F-PDA-DNA nanoprobes were washed three times with deionized water and dispersed in deionized water to obtain DTPA-modified [products / products]. 19 F-PDA-DNA nanoprobe solution;

[0072] Stirring at room temperature for 3 hours to add GdCl 3 Added to DTPA modification 19 Gd in F-PDA-DNA nanoprobe solution 3+ Complex, add NaOH to adjust the pH to 6.5, centrifuge at 10000 rpm and wash three times with water to remove unreacted Gd. 3+ Ultimately obtained 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0073] Reference Figure 1 This invention 19 Design diagram of F-PDA-DNA-DTPA / Gd nanoprobe Figure 2 This is a heatmap and electrophoresis diagram showing the base pairing probability. Figure 2 In the electrophoresis diagram, channel 1 is the marker, channel 2 is S1, channel 3 is S2, and channel 4 is S1S2. Single-stranded DNA S1 is ligated to a pre-synthesized... 19 The surface of F-PDA and hybridization with its complementary DNA S2 ( Figure 2 Subsequently, diethylenetriaminepentaacetic dianhydride (DTPA-DA) was bound to the 3' end of DNA S2, followed by the DTPA-modified... 19 F-PDA-DNA nanoprobes are generated by chelating Gd ions. 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0074] Figure 3 for 19 Transmission electron microscopy (TEM) image of F-PDA-DNA-DTPA / Gd nanoprobe, showing... 19 F-PDA-DNA-DTPA / Gd is a monodisperse spherical nanoprobe.

[0075] Figure 4 for 19 DLS analysis of F-PDA-DNA-DTPA / Gd nanoprobes showed that... 19The average particle size of F-PDA-DNA-DTPA / Gd is 110 nm. The circulation time of the nanoprobe in vivo depends on its size and shape, making this property ideal for biological and clinical applications.

[0076] The magnetic resonance imaging technique was used to evaluate 19 The in vitro response of F-PDA-DNA-DTPA / Gd to thrombin, the results are as follows: Figure 5 As shown, with the increase of thrombin concentration... 19 F and 1 The H-image signal showed a significant increase, indicating that thrombin binding led to the separation of complementary DNA strands, reaching its maximum activation state at a thrombin concentration of 4 μM. Initially, the signal was almost undetectable, but it increased significantly after incubation with thrombin.

[0077] In vitro cytotoxicity assessment

[0078] Cells were added to 96-well cell culture plates containing culture medium and incubated at 37°C for 24 hours under conditions of 5% carbon dioxide and 95% humidity to allow the cells to spread throughout the plate. The culture medium was then removed, and cells containing different concentrations of... 19 Incubate the cells in F-PDA-DNA-DTPA / Gd medium for 24 hours (note that the cell density in the 96-well plate must be greater than 70%). After washing twice with PBS, transfer the cells to 10% CCK-8 medium and incubate for another 2 hours. Finally, measure the absorbance at 450 nm using a microplate reader. Calculate the cell viability using the following formula.

[0079] Figure 6 HepG2 cells with different concentrations 19 Cell viability data after co-incubation with F-PDA-DNA-DTPA / Gd nanoprobes (CCK-8 assay results). As shown in the figure, cells exhibited high viability at all tested concentrations. However, no effect of the nanoparticles on cell proliferation and differentiation was observed. Even at the highest concentration (400 μg / mL), at least 90% of cells survived, highlighting the extremely low toxicity of the nanoprobes.

[0080] In vitro blood compatibility studies are another important indicator for assessing material toxicity and, to some extent, determine whether the material is suitable for in vivo application. We will use materials containing different concentrations of... 19Red blood cells were incubated with F-PDA-DNA-DTPA / Gd PBS buffer solution for 2 hours. It is well known that the intracellular osmotic pressure of red blood cells differs from that of the external environment in water. Red blood cells continuously absorb water and rupture. PBS buffer solution is similar to the internal environment of red blood cells, and red blood cells do not absorb water and rupture. The results, as shown in Figure 7, indicate that it has excellent blood compatibility. At a concentration of 400 μg / mL, red blood cells remained intact, with no significant difference compared to the negative control group. No ruptured red blood cells were detected in the supernatant. Calculations showed that the hemolysis rate of red blood cells was extremely low, indicating... 19 F-PDA-DNA-DTPA / Gd shows potential for in vivo application. Based on this, we further investigated... 19 Long-term toxicity of F-PDA-DNA-DTPA / Gd in mice. After intravenous injection of the nanomaterial, the mice remained healthy for one month, with a slight increase in weight. No abnormalities were observed in their diet, appearance, activity, exploratory behavior, urination, or nervous system, and they were no different from the control group.

[0081] Clinical blood routine examination such as Figure 8 The experimental group showed normal results compared to the control group.

[0082] Results of organ and tissue sections stained with hematoxylin and eosin (H&E) are as follows: Figure 9 The results showed 19 F-PDA-DNA-DTPA / Gd does not cause damage or inflammation to organs such as the heart, liver, spleen, lungs, and kidneys, ensuring good biocompatibility of this nanoprobe in vivo during diagnostic and therapeutic processes. Based on these results, we can conclude that... 19 F-PDA-DNA-DTPA / Gd showed no obvious adverse reactions in vivo and had good biocompatibility, indicating that the probe is suitable for use in vivo.

[0083] pass 19 f MRI test 19 The response of F-PDA-DNA-DTPA / Gd in live mice, specifically, 19 The F-PDA-DNA-DTPA / Gd probe was injected into the tumor of Balb / c mice, followed by the sequential injection of PBS (control group) and thrombin into the tumor. The results were as follows: Figure 10 As shown, 19 FRAM images showed a bright and distinct signal at the thrombin injection site, while no signal was observed at the PBS injection site. 1 H-MRI also showed significant signal enhancement. 19 The response of F-PDA-DNA-DTPA / Gd to thrombin is well-defined and significantly different from the response without a target.

[0084] Embedding the chemotherapy drug doxorubicin (DOX) 19 In F-PDA-DNA-DTPA / Gd, the -CG- bases of the DNA double strand provide loading sites for DOX. Fluorescence spectroscopy data indicate that without co-incubation with thrombin... 19 After centrifuging and collecting the supernatant of F-PDA-DNA-DTPA / Gd, the fluorescence spectrum was measured. No fluorescence intensity was observed, indicating that DOX was not released. However, after co-incubation with thrombin, the supernatant of PDA-DNA-DTPA / Gd was centrifuged and the fluorescence spectrum was measured, showing a certain fluorescence intensity, indicating that DOX was released after laser irradiation.

[0085] Subsequently, the supernatant from centrifuged samples after co-incubation with different concentrations of thrombin was measured for fluorescence spectra. Figure 11 As shown, the fluorescence intensity gradually increased with increasing thrombin concentration, indicating that 19F-PDA-DNA-DTPA / Gd can release more DOX with increasing co-incubation thrombin concentration. To verify the intracellular release of DOX, [further details are needed]. Figure 12 The results showed that no fluorescence was produced when the cells were co-incubated with PBS, indicating that the DNA double strands did not unwind and no DOX was released. However, red fluorescence was produced after co-incubation with thrombin, indicating that thrombin specifically binds to the aptamer, releasing DOX and thus producing a fluorescent signal.

[0086] Comparative Example 1

[0087] The difference between this comparative example and Example 1 is that the volume of 50 mg / mL DA aqueous solution added to the nanoemulsion suspension in step 1) was adjusted to 0.5 mL, 1 mL, and 1.5 mL, respectively. The remaining steps were the same as in Example 1, resulting in different amounts of DA aqueous solution used in this comparative example. 19 F-PDA-DNA-DTPA / Gd nanoprobe.

[0088] Figure 13 This comparative example uses different amounts of DA aqueous solution. 19 As can be seen from the F-PDA-DNA-DTPA / Gd nanoprobe, the probe morphology is not uniform under this condition, while Example 1 of the present invention can form a monodisperse uniform nanoprobe under a dosage of 2 ml.

[0089] In summary, this invention designs a 19The F-PDA-DNA-DTPA / Gd nanoprobe, acting as a responsive nanoparticle platform, is only visible upon activation by a specific biological trigger. Its biological applications are demonstrated through aptamer-targeted thrombin targeting and a strong and selective response to thrombin in live mice. Furthermore, the selected aptamer DNA is used for the first time in photothermal desorption, eliminating the need for enzymatic response; the probe itself dissociates via photothermal action, enabling the signal to be generated from nothing.

[0090] This invention uses 19 Preliminary demonstration of in vivo sensing using F-PDA-DNA-DTPA / Gd nanoprobes, combining 19 In the fields of fMRI, aptamer sensors, and molecular diagnostics, this provides a reference for developing diagnostic probes that will be activated in the presence of important disease-related analytes.

[0091] 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. A kind 19 The F-PDA-DNA-DTPA / Gd nanosensing probe is characterized by: It consists of an MR signal source, a quenching agent, and a response connector; The MR signal source is 19 F-PDA, wherein the quencher is Gd-DTPA, and the response linker is a DNA aptamer composed of a single DNA strand S1 and its complementary strand S2, with nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2; Among them, the 19 F-PDA was prepared according to the following method: 100 mg of cetyltrimethylammonium bromide was dispersed in 10–15 mL of water and ultrasonicated to obtain a dispersion. Add 55-65 μL of perfluorocarbon compound to the dispersion, and then sonicate at 50°C for 2 hours to obtain a nanoemulsion suspension. Add 90–110 μL of NH3·H2O and 25–35 mL of water to the nanoemulsion suspension, followed by 1–3 mL of a DA aqueous solution with a concentration of 45–55 mg / mL, and stir to obtain the desired solution. 19 F-PDA solution.

2. As described in claim 1 19 The F-PDA-DNA-DTPA / Gd nanosensing probe is characterized by: The ultrasonic treatment time for obtaining the dispersion is 30-60 minutes.

3. A device as described in claim 1 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: include, 19 F-PDA and single-stranded DNA S1 were reacted in Tris buffer with vigorous stirring, centrifuged and washed. The resulting product and 8-12 mM complementary DNA S2 were hybridized in PBS buffer, followed by centrifugation and washing to obtain... 19 F-PDA-DNA; 8-12 mmol of DTPA-DA dissolved in 19 A mixed solution was obtained by dissolving F-PDA-DNA in an aqueous solution. The pH of the mixed solution was adjusted, and DTPA-modified DNA was obtained by centrifugation. 19 F-PDA-DNA nanoprobes; DTPA modified 19 F-PDA-DNA nanoprobes were washed and dispersed in deionized water to obtain DTPA-modified [products / products]. 19 F-PDA-DNA nanoprobe solution; Stirring DTPA-modified [material] at room temperature 19 F-PDA-DNA nanoprobe solution, and GdCl3 was added to it for Gd... 3+ Complex, adjust pH, centrifuge and wash to remove unreacted Gd. 3+ Ultimately obtained 19 F-PDA-DNA-DTPA / Gd nanoprobe.

4. As described in claim 3 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: The 19 The reaction time of F-PDA with DNA single-strand S1 in Tris buffer with vigorous stirring is 10-14 h.

5. As described in claim 3 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: The reaction temperature for the hybridization reaction is 35~40℃.

6. As described in claim 3 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: The reaction time for the hybridization reaction is 1.5 to 2.5 hours.

7. As described in claim 3 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: The pH of the mixed solution is adjusted to 6.5-7.5 using NaOH.

8. As described in claim 3 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: The stirring of DTPA-modified [material] at room temperature 19 The stirring time for the F-PDA-DNA nanoprobe solution is 2.5~3.5h.

9. As described in claim 3 19 The method for preparing F-PDA-DNA-DTPA / Gd nanosensing probes is characterized by: After adjusting the pH, centrifugation and washing were performed to remove unreacted Gd. 3+ The pH should be adjusted to 6-7.

Images