BIOPROBE FOR NON-INVASIVE DIAGNOSIS OF PARKINSON'S DISEASE TRIGGERED BY INTESTINAL MICROENVIRONMENT, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

A bioprobe for a non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment, a preparation method thereof and use thereof are provided. The preparation method for the bioprobe provided by the present invention includes the following steps: (1) mixing europium nitrate with an organic ligand, and synthesizing a luminescent metal organic framework by a solvothermal method; (2) subjecting gold nanoparticles to a mixing reaction with aptamers to obtain Au-aptamer complexes; and (3) dissolving the luminescent metal organic framework in an Au-aptamer complex solution for a reaction, and washing the reaction product sequentially with deionized water, an ethanol solution, and a sodium dodecyl sulfate solution after the reaction to obtain the bioprobe. The present non-invasive oral bioprobe based on an intestinal microenvironment is configured for diagnosis of Parkinson's disease at an early stage.

Claims

1. A preparation method for a bioprobe for a non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment, comprising the following steps: (1) mixing europium nitrate with an organic ligand, and synthesizing a luminescent metal organic framework by a solvothermal method; (2) subjecting gold nanoparticles to a mixing reaction with aptamers to obtain Au-aptamer complexes; and (3) dissolving the luminescent metal organic framework in an Au-aptamer complex solution for a reaction to obtain a reaction product, and washing the reaction product sequentially with deionized water, an ethanol solution, and a sodium dodecyl sulfate solution after the reaction to obtain a luminescent metal organic framework adsorbing the Au-aptamer complexes, wherein the luminescent metal organic framework adsorbing the Au-aptamer complexes is the bioprobe.

2. The preparation method according to claim 1, wherein the europium nitrate and the organic ligand are in a molar ratio of (8-12):1.

3. The preparation method according to claim 2, wherein the organic ligand is one selected from the group consisting of 13,3-dihydroxy-2,2,5,5-tetramethyl-[1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid, [1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid, and [1,1:4,1:4,1-tetraphenyl]-3,3,5,5-tetracarboxylic acid.

4. The preparation method according to claim 3, wherein the solvothermal method is performed at a temperature of 160-200 C. for a period of 10-14 h.

5. The preparation method according to claim 4, wherein the mixing reaction is performed at a temperature of 25-37 C. at a rotation speed of 200-1000 rpm for a period of 3-5 h.

6. The preparation method according to claim 5, wherein the Au-aptamer complex solution is prepared with ultrapure water, and the Au-aptamer complex solution has a concentration of 0.5-5 mg/mL; the luminescent metal organic framework and the Au-aptamer complex solution are in a mass-to-volume ratio of (8-12) mg:(8-12) mL.

7. The preparation method according to claim 6, wherein the reaction in the step (3) is performed at a temperature of 25-37 C. for a period of 4-6 h.

8. A bioprobe for a non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment obtained by the preparation method according to claim 1.

9. A method of use of the bioprobe according to claim 8 in preparing a medicament for the non-invasive diagnosis of Parkinson's disease triggered by the intestinal microenvironment.

10. A method of use of the bioprobe according to claim 8 in preparing a medicament for a non-invasive detection of intestinal -synuclein triggered by an intestinal microenvironment.

11. The bioprobe according to claim 8, wherein in a process of preparing the bioprobe, the europium nitrate and the organic ligand are in a molar ratio of (8-12):1.

12. The bioprobe according to claim 11, wherein in the process of preparing the bioprobe, the organic ligand is one selected from the group consisting of 13,3-dihydroxy-2,2,5,5-tetramethyl-[1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid, [1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid, and [1,1:4,1:4,1-tetraphenyl]-3,3,5,5-tetracarboxylic acid.

13. The bioprobe according to claim 12, wherein in the process of preparing the bioprobe, the solvothermal method is performed at a temperature of 160-200 C. for a period of 10-14 h.

14. The bioprobe according to claim 13, wherein in the process of preparing the bioprobe, the mixing reaction is performed at a temperature of 25-37 C. at a rotation speed of 200-1000 rpm for a period of 3-5 h.

15. The bioprobe according to claim 14, wherein in the process of preparing the bioprobe, the Au-aptamer complex solution is prepared with ultrapure water, and the Au-aptamer complex solution has a concentration of 0.5-5 mg/mL; the luminescent metal organic framework and the Au-aptamer complex solution are in a mass-to-volume ratio of (8-12) mg:(8-12) mL.

16. The bioprobe according to claim 15, wherein in the process of preparing the bioprobe, the reaction in the step (3) is performed at a temperature of 25-37 C. for a period of 4-6 h.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1A-1B show TEM images of a europium-based acid-resistant luminescent metal organic framework and a bioprobe prepared in the examples;

[0027] FIGS. 2A-2B show confocal micrographs of a europium-based acid-resistant luminescent metal organic framework and a bioprobe prepared in Example 1;

[0028] FIG. 3 shows fluorescence spectra of -Syn at different concentrations in vitro;

[0029] FIG. 4 shows a standard curve of fluorescence intensity at 545 nm of -Syn at different concentrations in vitro;

[0030] FIGS. 5A-5B show fluorescence signals in the gastrointestinal tract of a Parkinson's disease mouse model, in which FIG. 5A shows mouse imaging, and FIG. 5B shows mouse intestinal tract imaging.

DETAILED DESCRIPTION

[0031] The present invention provides a preparation method for a bioprobe for non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment, which comprises the following steps: [0032] (1) mixing europium nitrate with an organic ligand, and synthesizing a luminescent metal organic framework by a solvothermal method; [0033] (2) subjecting gold nanoparticles to a mixing reaction with aptamers to obtain Au-aptamer complexes; and [0034] (3) dissolving the luminescent metal organic framework in an Au-aptamer complex solution for a reaction, and washing the reaction product sequentially with deionized water, an ethanol solution and a sodium dodecyl sulfate solution after the reaction, to obtain a luminescent metal organic framework adsorbing Au-aptamer complexes, namely the bioprobe.

[0035] In the present invention, when a bioprobe for non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment is prepared, europium nitrate is mixed with an organic ligand, and a luminescent metal organic framework is synthesized by a solvothermal method.

[0036] In the present invention, the europium nitrate and the organic ligand are preferably in a molar ratio of 8-12:1, and further preferably 10:1.

[0037] In the present invention, the organic ligand is preferably one of 13,3-dihydroxy-2,2,5,5-tetramethyl-[1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid, [1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid and [1,1:4,1:4,1-tetraphenyl]-3,3,5,5-tetracarboxylic acid, and further preferably 13,3-dihydroxy-2,2,5,5-tetramethyl-[1,1:4,1:4,1-quaterphenyl]-4,4-dicarboxylic acid.

[0038] In the present invention, the europium nitrate and the organic ligand are mixed in a DMF (N,N-dimethylformamide) solvent.

[0039] In the present invention, the europium nitrate, the organic ligand and the DMF are preferably in a molar-to-volume ratio of 8-12 mmol:1 mmol:8-12 mL, and further preferably 10 mmol:1 mmol:10 mL.

[0040] In the present invention, when the europium nitrate is mixed with the organic ligand, ultrasonic mixing is preferably adopted.

[0041] In the present invention, the ultrasonic mixing is preferably performed at a power of 100-1500 W, and further preferably 500 W.

[0042] In the present invention, the ultrasonic mixing is preferably performed for a period of 0.5-1.5 min, and further preferably 1 min.

[0043] In the present invention, the europium nitrate and the organic ligand are preferably synthesized in a 50 mL Teflon-lined stainless steel autoclave.

[0044] In the present invention, the solvothermal method is preferably performed at a temperature of 160-200 C., and further preferably 180 C.

[0045] In the present invention, the solvothermal method is preferably performed for a period of 10-14 h, and further preferably 12 h.

[0046] In the present invention, the luminescent metal organic framework preferably has an acid resistance range of pH 1.0-3.0, and further preferably pH 2.0; and preferably has a pore size of 30-40 , and further preferably 35 .

[0047] In the present invention, gold nanoparticles are subjected to a mixing reaction with aptamers to obtain Au-aptamer complexes.

[0048] In the present invention, the gold nanoparticles are preferably prepared as follows: an aqueous solution (2.5 mL) of 1 wt % trisodium citrate is added to a boiling aqueous solution (100 mL) containing 0.01 wt % HAuCl.sub.4, and immediately after addition, the solution is rapidly rotated (1000 rpm) in a flask, until the color of the solution gradually changes from gray to blue and then from purple to wine-red. Thereafter, the solution is allowed to boil under vigorous stirring (1000 rpm) for 10 min to verify the completion of the reaction. Finally, the solution is cooled to an ambient temperature (25 C.) and then stored at 4 C. for later use.

[0049] In the present invention, the aptamers are preferably aptamers of -Syn, with a nucleotide sequence of 5-SH-TTTTTGGTGGCTGGAGGGGGCGCGAACG, as shown in SEQ ID NO: 1, purchased from Sangon Biotech (Shanghai, China, https://www.sangon.com/). The purchased aptamer has been subjected to a sulfhydrylation treatment.

[0050] In the present invention, the gold nanoparticles and the aptamers are preferably in a molar concentration ratio of 0.5-1.5:1.5-2.5, and further preferably 1:2.

[0051] In the present invention, the mixing reaction of the gold nanoparticles and the aptamers is preferably performed at a temperature of 25-37 C., and further preferably 37 C.

[0052] In the present invention, the mixing reaction of the gold nanoparticles and the aptamers is preferably performed at a rotation speed of 200-1000 rpm, and further preferably 300 rpm.

[0053] In the present invention, the mixing reaction of the gold nanoparticles and the aptamers is preferably performed for a period of 3-5 h, and further preferably 4 h.

[0054] In the present invention, after the completion of the mixing reaction, the Au-aptamer complexes are further preferably washed.

[0055] In the present invention, the washing is preferably performed with a sodium dodecyl sulfate solution to remove unreacted aptamers.

[0056] In the present invention, the washing is preferably performed 2-4 times, and further preferably 3 times.

[0057] In the present invention, after each washing, centrifugal separation is preferably performed, followed by lyophilization at 40 C. to obtain Au-aptamer complexes.

[0058] In the present invention, the centrifugal separation is preferably performed at a rotation speed of 8000-12,000 rpm, and further preferably 10,000 rpm.

[0059] In the present invention, the centrifugal separation is preferably performed for a period of 8-12 min, and further preferably 10 min.

[0060] In the present invention, the luminescent metal organic framework prepared above is dissolved in an Au-aptamer complex solution for a reaction, and the reaction product is washed sequentially with deionized water, an ethanol solution and a sodium dodecyl sulfate solution to obtain a luminescent metal organic framework adsorbing Au-aptamer complexes, namely the bioprobe.

[0061] In the present invention, the Au-aptamer complex solution is preferably prepared with ultrapure water.

[0062] In the present invention, the Au-aptamer complex solution preferably has a concentration of 0.5-5 mg/mL, and further preferably 1 mg/mL.

[0063] In the present invention, the luminescent metal organic framework and the Au-aptamer complex solution are preferably in a mass-to-volume ratio of 8-12 mg:8-12 mL, and further preferably 10 mg:10 mL.

[0064] In the present invention, the reaction is preferably performed at a temperature of 25-37 C., and further preferably 37 C.

[0065] In the present invention, the reaction is preferably performed for a period of 4-6 h, and further preferably 5 h.

[0066] In the present invention, the ethanol solution preferably has a volume concentration of 70%.

[0067] In the present invention, the sodium dodecyl sulfate solution preferably has a concentration of 5% (W/W).

[0068] In the present invention, the washing is preferably performed for each reagent 3-5 times independently, and further preferably 4 times independently.

[0069] In the present invention, after the completion of the washing, the luminescent metal organic framework adsorbing Au-aptamer complexes is separated.

[0070] In the present invention, the separation is preferably centrifugal separation, followed by lyophilization at 40 C.

[0071] In the present invention, the centrifugal separation is preferably performed at a rotation speed of 8000-12,000 rpm, and further preferably 10,000 rpm.

[0072] In the present invention, the centrifugal separation is preferably performed for a period of 8-12 min, and further preferably 10 min.

[0073] The present invention also provides a bioprobe for non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment obtained by the preparation method.

[0074] The present invention also provides use of the bioprobe in preparing a medicament for non-invasive diagnosis of Parkinson's disease triggered by an intestinal microenvironment.

[0075] The present invention also provides use of the detection reagent in preparing a medicament for non-invasive detection of intestinal -synuclein triggered by an intestinal microenvironment.

[0076] The technical schemes provided by the present invention will be described in detail below with reference to the examples, which, however, should not be construed as limiting the scope of the present invention.

Example 1

[0077] (1) Preparation of a porous acid-resistant luminescent metal organic framework: Eu(N03).sub.3 (136 mg, 0.3 mmol) and 13,3-dihydroxy-2,2,5,5-tetramethyl-[1,1:4,1:4,1-tetrabiphenyl]-4,4-dicarboxylic acid (43.5 mg, 0.03 mmol) were dispersed in 10 mL of DMF, and the suspension was sonicated at 500 W for 1 min. The resulting solution was then transferred to a 50 mL Teflon-lined stainless steel autoclave and subjected to a heat treatment at 180 C. for 12 h, so that a luminescent metal organic framework with porous and acid resistant properties was obtained. The obtained luminescent metal organic framework was dried in a glove box at 80 C. for 4 h. The luminescent metal organic framework was determined to have an acid resistance value of pH 1.0-3.0 and a pore size of 35 . [0078] (2) Preparation of Au-aptamers: an aqueous solution (2.5 mL) of 1 wt % trisodium citrate was added to a boiling aqueous solution (100 mL) containing 0.01 wt % HAuCl.sub.4, and immediately after addition, the solution was rapidly rotated (1000 rpm) in a flask, until the color of the solution gradually changed from gray to blue and then from purple to wine-red. Thereafter, the solution was allowed to boil under vigorous stirring (1000 rpm) for 10 min to verify completion of the reaction, so that gold nanoparticles were obtained. Finally, the solution was cooled to an ambient temperature (25 C.) and then stored at 4 C. for later use.

[0079] The aptamers were aptamers of -Syn, with a nucleotide sequence of 5-SH-TTTTTGGTGGCTGGAGGGGGCGCGAACG, as shown in SEQ ID NO: 1, purchased from Sangon Biotech (Shanghai, China, https://www.sangon.com/).

[0080] A 1 M gold nanoparticle solution (prepared with ultrapure water) was mixed with a 2 M aptamer solution (prepared with ultrapure water), and the mixture was slowly rotated (300 rpm) at 37 C. for 4 h, subjected to centrifugal separation (10,000 rpm, 10 min), and washed with ultrapure water 3 times, each followed by centrifugal separation (10,000 rpm, 10 min), so that unreacted aptamers were removed by washing and centrifugation. Finally, the solid obtained by the last centrifugal separation was lyophilized to obtain Au-aptamers, which were stored at 4 C. for later use. [0081] (3) Modification of the luminescent metal organic framework with the Au-aptamers: an Au-aptamer solution at a concentration of 1 mg/mL was prepared with ultrapure water as a solvent. The dried luminescent metal organic framework (10 mg) was quickly taken out of the glove box and dissolved in 10 mL of the Au-aptamer solution at room temperature (25 C.) for 5 h of reaction. Then, the mixture was centrifuged (10,000 rpm, 10 min), washed sequentially with deionized water, a 70% ethanol solution and a 5% (W/W) sodium dodecyl sulfate solution, each for 4 times (SDS can remove aptamers adsorbed on the surface of the luminescent metal organic framework so as to only reserve aptamers inside its pores), and subjected to centrifugal separation (10,000 rpm, 10 min), so that the bioprobe with the Au-aptamers adsorbed inside the luminescent metal organic framework.

[0082] The luminescent metal organic framework prepared in the step (1) exhibited a one-dimensional linear structure by TEM, as shown in FIG. 1A. After the modification with the Au-aptamers, as shown in FIG. 1B, individually separated gold nanoparticles can be seen clearly in the luminescent metal organic framework from FIG. 1B, suggesting that the Au-aptamers can be accurately loaded into the cavity of the luminescent metal organic framework to form a bioprobe. FIGS. 2A-2B show confocal micrographs of the luminescent metal organic framework (FIG. 2A) and the bioprobe (FIG. 2B). It shows that the luminescent metal organic framework can exhibit green fluorescence. However, when the aptamers modified on the surface of the gold nanoparticles were effectively loaded into the cavity of the luminescent metal organic framework, the green fluorescence exhibited by the luminescent metal organic framework was quenched through fluorescence resonance energy transfer of the gold nanoparticles, so that the non-luminescent bioprobe was obtained. This further confirms the formation of the bioprobe.

[0083] The bioprobe prepared above was used for in vitro detection and the fluorescence spectra and a standard curve of -Syn at different concentrations were plotted. -Syn at different concentrations was dissolved in PBS to react with 1 mg/mL bioprobe for 30 min. The solution after the reaction was measured for its fluorescence intensity by the FL-4600 molecular fluorometer. The results are shown in FIGS. 3 and 4. As can be seen from the figures, the bioprobe provided by the present invention can realize an in vitro -Syn detection with high detection sensitivity and good standard curve linearity.

[0084] The present invention also constructs a Parkinson's disease mouse model for an in vivo experiment.

[0085] Construction of Parkinson's Disease Mouse Model:

[0086] MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) has high lipid solubility, is easy to permeate the blood-brain barrier, and can be converted into an effective component MPP.sup.+ under the action of glial cell monoamine oxidase after entering the brain. MPP.sup.+ can inhibit the activity of mitochondrial complex I and lead to the degeneration and death of dopaminergic neurons after it is taken into the mitochondria of dopaminergic neurons by dopamine transporters.

[0087] 8-week-old male mice C57BL/6 were intraperitoneally injected with MPTP (0.6 mg, 2 mg/mL) daily for 7 consecutive days to obtain mice with Parkinson's disease.

[0088] Then, in the mice with Parkinson's disease, after oral administration of the bioprobe (at a dose of 50 mg/kg), fluorescence signals to the gastrointestinal tract were detected by a small animal imager, as shown in FIGS. 5A-5B. This suggests that the bioprobe triggered by the intestinal microenvironment can respond to the -Syn of the gastrointestinal tract. Meanwhile, the bioprobe triggered by the intestinal microenvironment can be retained along with the feces. The content of -Syn in the feces of mice was quantitatively determined and compared with that obtained using a commercial ELISA kit, and the results are shown in Table 1.

TABLE-US-00001 TABLE 1 Comparison of the results of -Syn concentration in feces obtained using the bioprobe of Example 1 vs. a commercial ELISA kit (x s, n = 6) Enzyme-linked Additional immunosorbent Background concentration assay kit Bioprobe Recovery Sample (g/mL) (g/mL) (g/mL) (g/mL) (%) 1 2.0 0.050 2.059 0.011 2.051 0.001 102.00 2 0.4 0.100 0.495 0.011 0.498 0.009 98.00 3 0 0.500 0.490 0.050 0.493 0.091 98.60 4 0 1.000 0.980 0.040 0.982 0.060 98.20 5 0.3 5.000 5.310 0.250 5.310 0.150 100.20 6 5 10.000 14.508 0.312 14.489 0.249 94.89

[0089] As can be seen from Table 1, the detection result of the developed non-invasive oral bioprobe for the intestinal microenvironment on the feces of mice with Parkinson's disease was consistent with the result obtained by the commercial enzyme-linked immunosorbent assay kit, demonstrating the reliability of the results.

[0090] According to the above examples, the present invention provides a non-invasive oral bioprobe based on an intestinal microenvironment, for the purpose of diagnosis of Parkinson's disease at an early stage. This provides a brand new scheme for oral bioprobes, and solves the problem that the oral bioprobes cannot be stably present in the gastrointestinal tract. Moreover, the method is suitable for patients to test themselves at home, thereby bringing great convenience to the patients. The method opens up a new approach for early non-invasive diseases.

[0091] The above descriptions are only preferred embodiments of the present invention. It should be noted that those of ordinary skill in the art can also make several improvements and modifications without departing from the principle of the present invention, and such improvements and modifications shall fall within the protection scope of the present invention.