Tumor microacid-glutathione cascade response nanoprobe and its preparation method and application

Abstract

The present invention discloses an ultra-sensitive nanoprobe based on tumor microacid and glutathione (GSH) cascade response type and its preparation method and application, which is composed of microacid response matrix material and GSH activated near-infrared cyanine dye. The probe of the present invention has high specificity due to dual marker targeting, and its detection sensitivity is greatly improved due to the cascade response design. Compared with traditional nano-imaging probes and small molecule dyes, it can reduce imaging false positive signals on the one hand, and greatly reduce biological tissue background signals on the other hand, realize high-sensitivity and high-specificity fluorescence imaging of lesion tissue, and provide a method for accurate diagnosis and real-time detection of tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular imaging, and in particular relates to a tumor microacid and glutathione (GSH) cascade response type ultra-sensitive nanoprobe and a preparation method and application thereof. Background Art

[0002] Molecular imaging technology uses imaging methods to display specific molecules at the tissue level, cell level and subcellular level, reflecting the changes in the molecular level in the living state, and realizing real-time, non-invasive and dynamic imaging of in vivo physiology and pathology. It can be used to study the occurrence, development and metastasis of living diseases or tumors. Near-infrared fluorescence imaging has great potential in the field of tumor diagnosis due to its advantages of non-invasiveness, no radiation, rapid imaging and high sensitivity.

[0003] The core of molecular imaging technology is molecular imaging probes. However, most of the current near-infrared fluorescent probes are always-ON. After entering the body, the probes will circulate throughout the body with the blood, generating strong background signals, which not only have a low imaging signal-to-noise ratio, but also easily generate false positive signals. The responsive (OFF-ON) probes do not emit light in normal tissues and blood, and are only lit up when they reach the tumor site to generate fluorescent signals, which can significantly reduce the background signal from the probe itself, thereby improving the imaging sensitivity and resolution and reducing the generation of false positive signals. Therefore, by constructing a responsive near-infrared fluorescent imaging probe so that its fluorescent signal can be activated by a specific tumor-related marker, it is possible to achieve highly sensitive and highly specific visual tracking detection of the target tissue. However, most pathological parameters and biological targets are usually shared by multiple types of cells. These markers are not only overexpressed in lesion tissues, but also expressed in normal cells and blood. The current single-target activation probes may have the problem of non-specific activation under complex physiological and pathological environments, resulting in false positive signals in imaging.

[0004] For these reasons, developing imaging probes with multi-target synergistic activation can effectively avoid false positive signals caused by nonspecific activation. Constructing highly specific imaging probes that meet the needs of tumor detection is both an urgent clinical need and a research hotspot in the biomedical field. Summary of the invention

[0005] In order to improve the above technical problems, the present invention provides a nanoprobe, comprising a slightly acid-responsive matrix and a GSH-activated near-infrared cyanine dye;

[0006] According to an embodiment of the present invention, the GSH-activated near-infrared cyanine dye is Cy7SS (Heptamethinecyanine dyes-disulfide), and the Cy7SS has a structure shown in the general formula Cy7SS-I or Cy7SS-II:

[0007]

[0008] Where: X is one of C(CH3)2, NCH3, O, S, Se; Y is a halogen ion, PF6 － or TsO － One of the following; R1 is C 1-6 Alkyl, CH2C6H5, (CH2) n COOH and (CH2) n COO - One of the following, n is an integer from 2 to 6; R2 is one of H, SO3R3, OH or halogen; R3 is NH(CH2)2SS(CH2)2NH2, NH(CH2)2SeSe(CH2)2NH2 or NH(CH2)2SSC5H4N.

[0009] According to an embodiment of the present invention, X is preferably C(CH3)2; Y is preferably I - ; The R1 is preferably C2H5; The R2 is preferably H; The R3 is preferably NH(CH2)2SS(CH2)2NH2.

[0010] According to an embodiment of the present invention, the structure of the Cy7SS is as follows:

[0011]

[0012] According to an embodiment of the present invention, the slightly acid-responsive matrix is ​​calcium phosphate (abbreviated as CaP); in some embodiments, the calcium phosphate is in an amorphous form; preferably, the amorphous form is Ca3(PO4)2·xH2O, where x is 0, 1, 2, 3, 4 or 5.

[0013] According to an embodiment of the present invention, the calcium phosphate can be obtained by the following reaction: by using polyacrylic acid and calcium salt to self-assemble in isopropanol and then mineralize and precipitate under the action of phosphate; the calcium salt can be calcium hydroxide; the phosphate can be diammonium hydrogen phosphate. The carboxyl group of polyacrylic acid can coordinate with the calcium ion in the calcium salt, and further mineralize and precipitate under the action of phosphate to form the calcium phosphate.

[0014] According to an embodiment of the present invention, the GSH-activated near-infrared cyanine dye is coated on the slightly acid-responsive matrix.

[0015] According to an embodiment of the present invention, the slightly acid-responsive matrix has an ultrasensitive response range that matches the slightly acidic environment of the tumor, and the response pH range is 7.2 to 6.5, for example, the response pH range is 7.05 to 6.75; ΔpH can be less than 0.3, for example, 0.2; exemplary response pHs are 7.0, 6.9, 6.8, 6.5, and 6.25.

[0016] The present invention also provides a method for preparing the nanoprobe (Cy7SS@CaP), comprising the following steps:

[0017] (1) Preparation of GSH-activated near-infrared cyanine dye Cy7SS-I or Cy7SS-II: Compound Cy7SS-Ia reacts with R3H to obtain Cy7SS-I; or, compound Cy7SS-IIa reacts with R3H to obtain Cy7SS-II;

[0018]

[0019]

[0020] (2) assembling and encapsulating the Cy7SS-I or Cy7SS-II obtained in step (1) in a slightly acid-responsive matrix to synthesize a Cy7SS@CaP nano-imaging probe;

[0021] Wherein, X, Y, R1, R2, R3, and the slightly acidic responsive matrix have the definitions described above; X1 is selected from Cl, Br or I, preferably Cl;

[0022] According to an embodiment of the present invention, in step (1), the exposed amino group in R3H undergoes a substitution reaction with position X1 in Cy7SS-Ia or Cy7SS-IIa;

[0023] According to an embodiment of the present invention, in step (1), the R3H is, for example, NH2(CH2)2SS(CH2)2NH2, NH2(CH2)2SeSe(CH2)2NH2 or NH2(CH2)2SSC5H4N;

[0024] According to an embodiment of the present invention, in step (1), the reaction can be carried out in the presence of an organic solvent, and the solvent is at least one of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (MeCN), dichloromethane (DCM), and chloroform, preferably DMF;

[0025] According to an embodiment of the present invention, in step (1), the reaction can be carried out under the action of a catalyst, and the catalyst can be an organic base or an inorganic base, such as triethylamine, diisopropylethylamine (DIPEA), pyridine, piperidine, sodium carbonate, potassium carbonate, sodium bicarbonate, preferably triethylamine;

[0026] According to an embodiment of the present invention, in step (1), the reaction temperature is 25-45°C, exemplarily 40°C; the reaction time is 12-36h, for example 24h.

[0027] According to an embodiment of the present invention, in step (2), the assembly and coating method is: mixing calcium polyacrylate, Cy7SS and diammonium hydrogen phosphate in a solvent to obtain the nanoprobe;

[0028] According to an embodiment of the present invention, the solvent may be a mixed solvent of water and an organic solvent, and the organic solvent may be at least one of methanol, ethanol, and isopropanol;

[0029] According to an embodiment of the present invention, in step (2), the assembly coating method is: dissolve 20% polyacrylic acid in deionized water, add calcium hydroxide, stir to dissolve, dissolve Cy7SS in DMSO and drip into the above reaction solution, then add isopropanol and diammonium hydrogen phosphate, stir for 8-24 hours, and obtain the nanoprobe.

[0030] According to an exemplary embodiment of the present invention, in step (2), the assembly and coating method is: dissolve 50-90 μL of 20% polyacrylic acid (PAA, molecular weight 2000-5000) in 10 mL of deionized water, add 5-8 mg of calcium hydroxide, stir to dissolve, dissolve different dyes Cy7SS in 100-500 μL of DMSO, drop into the above liquid and stir for 30 minutes, slowly add 10-50 mL of isopropanol to the above reaction solution, add 5-10 mg of diammonium phosphate, stir for 8-24 hours, centrifuge and wash with water 3 times, disperse in deionized water to obtain the nanoprobe.

[0031] The present invention also provides the use of the nanoprobe in tumor detection, for example, in vivo tumor imaging. According to an embodiment of the present invention, the tumor is selected from breast cancer.

[0032] The present invention also provides a method for using the nanoprobe for in vivo tumor imaging: the probe is injected into a mouse through the tail vein, and the probe is aggregated at the tumor site through EPR (enhanced permeability and retention effect). In normal tissues and blood, the nanoparticles are in a quenched state due to being double-locked; the micro-acid metabolism at the tumor site causes the micro-acid response matrix material (i.e., CaP) to disintegrate, and then the dye Cy7SS enters the cell and is activated by GSH, and the fluorescent signal is cascade-amplified after continuous activation by tumor micro-acid and GSH, thereby achieving highly sensitive and accurate detection of tumors.

[0033] Beneficial Effects

[0034] Through the above technical scheme, the present invention provides a tumor microacid, GSH cascade response type ultra-sensitive nanoprobe and its preparation method and application. Compared with the prior art, its technical advantages are:

[0035] 1. The present invention combines tumor microacid-activated matrix and GSH-activated dye, and uses double locking to quench fluorescence, giving the nanoprobe more excellent performance. The nano-imaging probe quenches near-infrared luminescence through dye modification, and then further quenches by assembling and aggregating to form CaP nanoparticles. Double quenching greatly reduces background fluorescence, which is helpful for high signal-to-noise ratio imaging of tumors.

[0036] 2. The nanoprobe provided by the present invention responds to the dual markers of microacid and GSH in sequence for cascade amplification, which effectively improves the problems of poor specificity and low signal-to-noise ratio in biological imaging of existing fluorescent imaging probes, improves the specificity of tumor detection, and reduces the false positives in the imaging process caused by single-target contrast agents.

[0037] 3. The nanoprobe provided by the present invention has a slightly acidic and GSH cascade amplification effect, improved detection sensitivity, simple synthesis, excellent optical properties, and good biocompatibility, and can be used for ultrasensitive detection of tumors in vivo.

[0038] 4. The nanoprobe provided by the present invention has an ultra-sensitive response range that matches the slightly acidic environment of the tumor, and the response pH range is 7.2 to 6.5, which greatly expands the response range of the tumor detection probe; and it can sensitively respond to slight pH changes, and the change range of ΔpH is at least 0.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Cy7SS fluorescence spectrum for implementation example 1

[0040] Figure 2 Cy7SS responsive fluorescence spectroscopy described in Example 2

[0041] Figure 3TEM image of Cy7SS@CaP described in Example 3

[0042] Figure 4 Acid response curve of Cy7SS@CaP described in Example 4

[0043] Figure 5 Cy7SS@CaP cascade responsive spectroscopy for implementation example 4

[0044] Figure 6 For the implementation of the nano-imaging probe cytotoxicity described in Example 5

[0045] Figure 7 For implementing the nano-imaging probe described in case 6 for breast cancer detection

[0046] Definition and explanation of terms

[0047] Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, preferred definitions, definitions of specific compounds in the embodiments, etc., can be arbitrarily combined and combined with each other. The group definitions and compound structures after such combination and combination shall fall within the scope of the description of this application.

[0048] Unless otherwise indicated, the subscripts of groups in this application generally refer to the number of the group.

[0049] When the numerical range described in the specification and claims of this application is defined as "integer", it should be understood that the two endpoints of the range and each integer in the range are recorded. For example, "integers from 0 to 6" should be understood as recording each integer of 0, 1, 2, 3, 4, 5 and 6. When the numerical range is defined as "number", it should be understood as recording the two endpoints of the range, each integer in the range and each decimal in the range.

[0050] The term "halogen" refers to F, Cl, Br, and I. In other words, F, Cl, Br, and I may be described as "halogen" in the present specification.

[0051] The present invention uses "C" alone or as a suffix or prefix. 1-6 "Alkyl" means branched and straight chain saturated aliphatic hydrocarbon groups having from 1 to 6 carbon atoms (or a specific number of carbon atoms if that number is provided). 1-6Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, 1,2-dimethylbutyl, 2-ethylhexyl, 3-ethylhexyl, 2-hexyldecyl, and the like, as well as all isomeric forms of the foregoing groups. DETAILED DESCRIPTION

[0052] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0053] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0054] In order to solve the problems of low signal-to-noise ratio and poor specificity in the prior art, the specific technical solution adopted by the present invention is: using the weakly acidic microenvironment of tumors and the highly expressed GSH in tumor cells as dual targets, further quenching by encapsulating the GSH-activatable near-infrared cyanine dye in a slightly acid-responsive matrix material, and designing and synthesizing a slightly acid-GSH cascade-responsive fluorescent imaging probe. The nano-imaging probe of the present invention improves the detection specificity through the dual targeting of slightly acid and GSH, amplifies the imaging signal through continuous activation of the cascade response, and improves the detection sensitivity, thereby meeting the needs of tumor detection.

[0055] Implementation Case 1

[0056] The fluorescent dye part of the cascade response near-infrared nano-imaging probe is modified with heptamethine cyanine dye, and the general formula is Cy7SS-I and Cy7SS-II. The preparation process is introduced by taking the following reaction equation as an example.

[0057]

[0058] Step 1: Reaction of Cy7 and cystamine dihydrochloride to prepare Cy7SS: Weigh 200.0 mg Cy7, 337.68 mg cystamine dihydrochloride, and 207.315 mg potassium carbonate, dissolve in 5 mL DMF, react at 40° C. for 24 h under argon protection, and the solution turns dark blue.

[0059] Step 2: Isolation and purification of Cy7SS-1: The product obtained in step 1 was precipitated with ether, the developing solvent ratio was adjusted, and it was separated by column chromatography, the blue segment was collected, and the compound Cy7SS-1 was obtained by rotary evaporation.

[0060] Fluorescence spectrum test of Cy7SS-1:

[0061] Take 1 mg of Cy7SS-1 and dissolve it in 1 mL of DMSO to prepare a stock solution with a concentration of 1.25×10 -3 mol / L; 80 μL of the mother solution was added to a 5 mL centrifuge tube and methanol was added to prepare a concentration of 2.5×10 -5 mol / L of the test solution; the spectrum was tested at room temperature with an excitation wavelength of 640nm. The results are as follows Figure 1 As shown, the maximum emission peak is located at 745 nm.

[0062] Implementation Case 2

[0063] In order to investigate the GSH response performance of Cy7SS-I and Cy7SS-II, the relationship between the absorption spectrum, emission spectrum and GSH concentration was tested by taking Cy7SS-1 as an example.

[0064] Step 1: Prepare the dye stock solution: Dissolve 1 mg of Cy7SS-1 in 1 mL of DMSO to prepare the dye stock solution at a concentration of 1.25 × 10 -3 mol / L; weigh 30.7 mg GSH and dissolve it in 1 mL deionized water to prepare 100 mM GSH stock solution, dilute to 10 mM and 1 mM for later use.

[0065] Step 2: Prepare working solution: Take 21 2 mL centrifuge tubes, number them 1-7, and add 1.7 mL, 1.8 mL, 1.86 mL, 1.7 mL, 1.8 mL, 1.86 mL, 1.9 mL of DMSO, 200 μL, 100 μL, 40 μL of 10 mM GSH stock solution, 200 μL, 100 μL, 40 μL, 0 μL of 1 mM GSH stock solution, and 100 μL of Cy7SS-1 stock solution to make the working solution dye concentration 6.25 × 10 -5 mol / L, the GSH concentrations of samples 1-7 were 1mM, 500μM, 200μM, 100μM, 50μM, 20μM and 0μM, and their fluorescence emission spectra were measured.

[0066] The results of the test are as follows Figure 2As shown in the figure, as the GSH concentration increases from 0μM to 1mM, the fluorescence at 820nm gradually increases from zero to one, showing very good response performance. The experimental results show that Cy7SS-1 is sensitive to GSH and is related to concentration changes. The detection limit is at least 20μM, which can well match the GSH concentration in the tumor area and can be used for high-sensitivity tumor detection.

[0067] Implementation Case 3:

[0068] In order to achieve the dual quenching effect of the present invention, the obtained Cy7SS-1 is coated in a slightly acid-responsive matrix by self-assembly to achieve further quenching of the fluorescence signal. Taking Cy7SS-1 as an example of a GSH-activated dye and CaP as an example of a slightly acid-responsive matrix, the preparation process of the cascade response nano-imaging probe is introduced.

[0069] Use a pipette to accurately measure 50μL 20% PAA (Mw = 2000), dissolve it in 10mL deionized water under ultrasonic conditions, add 7.5mg calcium hydroxide, ultrasonically dissolve it, place it on a magnetic stirrer, add 1mg Cy7SS-1 under argon protection, and stir at room temperature for 30min. Slowly drip 50mL of isopropanol to form a milky white solution, and the addition is completed in 30min. Accurately weigh 9.6mg of diammonium hydrogen phosphate and add it to the above reaction solution, and stir at room temperature for 24h. After the reaction is completed, collect the product by centrifugation, wash it 3 times with 50mL deionized water, collect the centrifugal product and disperse it in 5mL deionized water, which is the cascade response nano-imaging probe Cy7SS@CaP.

[0070] Referring to the above steps, replacing Cy7SS-1 with Cy7, the slightly acid-responsive control probe Cy7S@CaP can be prepared.

[0071] In order to investigate the size and morphology of the nano-imaging probe, the Cy7SS@CaP nano-probe was characterized by transmission electron microscopy (TEM). Figure 3 As shown, the nanoprobes are uniform in size and well dispersed, with a diameter of about 100 nm.

[0072] Implementation Case 4:

[0073] In order to investigate the response sensitivity of the cascade response nanoprobe, Cy7SS@CaP was taken as an example to test its fluorescence emission spectrum at different pH values ​​and its spectral changes under sequential activation by slight acid and GSH.

[0074] Step 1: Prepare the mother solution: Disperse the prepared Cy7SS@CaP in 1 mL of deionized water at a concentration of 50 mg / mL.

[0075] Step 2: Prepare working solution and test slight acid responsiveness: Take 27 2mL centrifuge tubes, number them 1-9, and add 1.8mL sodium citrate buffer solution with pH values ​​of 7.4, 7.25, 7.05, 6.9, 6.8, 6.5, 6.25, 6.05, and 5.85 to three groups of parallel samples, respectively. Add 200μL Cy7SS@CaP mother solution to each tube and measure its fluorescence emission spectrum (channel 1). The results are shown in Figure 2. Figure 4 As shown, the fluorescence signal can sensitively respond to slight pH changes, with a ΔpH of 0.2.

[0076] Step 3: Prepare working solution and test cascade responsiveness: Take 9 2mL centrifuge tubes, number them 1-3, and divide them into 3 parallel groups. Dilute 200μL Cy7SS@CaP stock solution in 2.0mL sodium citrate buffer solution with pH 7.4, 2.0mL sodium citrate buffer solution with pH 6.8, and 1.8mL sodium citrate buffer solution with pH 6.8, respectively. Then, add 200μL 10mM GSH stock solution and shake. Test the fluorescence spectra respectively. The results are as follows: Figure 5 As shown, the fluorescence signal at 820 nm undergoes continuous activation of the pH and GSH dual markers, and the signal cascade is amplified.

[0077] Implementation Case 5:

[0078] Good biocompatibility is the basis of in vivo imaging detection of nano-imaging probes. For this purpose, Cy7SS@CaP was taken as an example to verify its cytotoxicity to breast cancer cell lines.

[0079] 4T1 cell line was used, and cells were cultured in DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin in an incubator at 37°C and 5% carbon dioxide. 4T1 cells were cultured to the logarithmic growth phase and 1×10 4 The density of cells / well was transferred to a 96-well plate to ensure that the cells were evenly dispersed and cultured overnight in a constant temperature incubator. Different concentrations of Cy7SS@CaP were added to the 96-well plate, and a blank control group with no cells but only culture medium was set up. Six replicate wells were set up for each concentration, and the cells were incubated for 24 hours to perform the MTT experiment. The cell survival rate was calculated as follows: (average absorption value of the experimental group / average absorption value of the control group) × 100%. The final result was obtained by calculating the average and standard deviation of the six parallel experiments in each group, as shown in Figure 2. Figure 6 shown.

[0080] Implementation Case 6:

[0081] In order to investigate the detection performance of cascade response nano-imaging probes for tumors at the in vivo level, taking Cy7SS@CaP as an example, the single response probe and the always on control probe were selected to image the breast cancer mouse model.

[0082] Several tumor-bearing mice with similar physiological states and tumor volumes were selected and divided into 4 groups on average. Nano-imaging probes were injected into the tail vein, including cascade response probe Cy7SS@CaP, slightly acid response control probe Cy7S@CaP, GSH response control probe SiO2-Cy7SS, and always on control probe SiO2-Cy7S. After the probe injection, the fluorescence signal of the mouse at 790nm was collected using a small animal in vivo imaging system at different time points. The fluorescence intensity of the tumor site at each time point was compared among the groups, and the signal-to-noise ratio was calculated at the highest signal point. The results are shown in the figure. Figure 7 The results show that the imaging performance of the cascade response nano-imaging probe is better than that of the traditional imaging probe, and the signal-to-noise ratio is much greater than that of the control group, which can sensitively detect tumor lesions.

[0083] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanoprobe comprising a slightly acid-responsive matrix and a GSH-activated near-infrared cyanine dye; The GSH-activated near-infrared cyanine dye is Cy7SS, and the Cy7SS has a structure shown in the general formula Cy7SS-I: in: The X is C(CH3)2; the Y is I - ; R1 is C2H5; R2 is H; R3 is NH(CH2)2SS(CH2)2NH2; The slightly acidic response matrix is ​​calcium phosphate; The GSH-activated near-infrared cyanine dye is coated on the slightly acid-responsive matrix.

2. The method for preparing the nanoprobe according to claim 1, characterized in that: The steps include: (1) Preparation of GSH-activated near-infrared cyanine dye Cy7SS-I: Compound Cy7SS-Ia was reacted with R3H to obtain Cy7SS-I; (2) assembling and encapsulating the Cy7SS-I obtained in step (1) in a slightly acid-responsive matrix to synthesize a Cy7SS@CaP nano-imaging probe; Wherein, X, Y, R1, R2, R3, and the slightly acidic responsive matrix have the definitions as described in claim 1; X1 is selected from Cl, Br or I; In step (1), the exposed amino group in R3H undergoes a substitution reaction with position X1 in Cy7SS-Ia; In step (1), the reaction is carried out in the presence of an organic solvent, wherein the solvent is at least one of N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, dichloromethane and chloroform; In step (1), the reaction is carried out under the action of a catalyst, and the catalyst is an organic base or an inorganic base; In step (1), the reaction temperature is 25-45°C; the reaction time is 12-36h; In step (2), the assembly coating method is: dissolving 20% ​​polyacrylic acid in deionized water, adding calcium hydroxide, stirring to dissolve, dissolving Cy7SS in DMSO and dripping into the above reaction solution, then adding isopropanol and diammonium hydrogen phosphate, stirring for 8-24 hours, to obtain the nanoprobe.

3. The preparation method according to claim 2, characterized in that: The catalyst is selected from triethylamine, diisopropylethylamine, pyridine, piperidine, sodium carbonate, potassium carbonate, and sodium bicarbonate.

4. The preparation method according to claim 2, characterized in that: In step (2), the assembly and coating method is as follows: dissolve 50-90 μL of 20% polyacrylic acid in 10 mL of deionized water, add 5-8 mg of calcium hydroxide, stir to dissolve, dissolve the dye Cy7SS in 100-500 μL of DMSO, drop it into the above reaction solution and stir for 30 minutes, slowly add 10-50 mL of isopropanol, add 5-10 mg of diammonium phosphate, stir for 8-24 hours, centrifuge and wash with water 3 times, disperse in deionized water to obtain the nanoprobe.

5. Use of the nanoprobe according to claim 1 in the preparation of drugs for tumor detection.

6. The use according to claim 5, characterized in that: The drug is a drug used for in vivo tumor imaging.

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