A multi-layer organic metal frame-nano gold mediated aptamer functionalized magnetic nano probe and a preparation method thereof

Abstract

The application discloses a kind of based on multilayer organic metal frame-nano gold mediated aptamer functionalized magnetic nano probe and preparation method thereof, the aptamer functionalized magnetic nano probe is with the magnetic nanoparticle of multilayer organic metal frame-nano gold composite material in situ growth on polydopamine coating and it is carrier, high-efficiency bonding aptamer fluorescence probe is formed by using mercapto reagent blocking;The aptamer fluorescence probe is formed by the nucleic acid aptamer DNA chain with 5' end with mercapto and 5' end with fluorescent label group cDNA complementary strand hybridization;The multilayer organic metal frame-nano gold composite material is by the lysine amino UIO-66 with multilayer structure and nano gold bonding composition.The aptamer functionalized magnetic nano probe prepared in the application surface amino is rich, can high-efficiency bonding nano gold and bridge connection aptamer fluorescence probe, with the advantages such as high binding efficiency with target substance, strong recognition specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, and provides a kind of based on multilayer organic metal framework-nano gold mediated aptamer magnetic nano probe and preparation method thereof. BACKGROUND

[0002] Okadaic acid is a common red tide algal toxin, which is enriched in the digestive glands of various marine mollusks. Consuming seafood contaminated with okadaic acid can cause diarrhea poisoning. Therefore, it is of great significance to carry out okadaic acid monitoring to protect food safety and consumer health. Currently, the monitoring techniques for okadaic acid mainly include chromatography, sensor method and biological method, etc. In the field of sensor analysis, the analysis techniques using aptamer as recognition element have attracted widespread attention. Aptamer has the advantages of good affinity, easy separation and stable signal, etc. The functionalized magnetic nanoprobe prepared by coupling aptamer with magnetic nanoparticles is a new type of targeted nanoprobe, which has been rapidly developed in the selective extraction, separation and detection of red tide algal toxins. In the research of aptamer nanoprobe recognition technology, how to construct a stable and specific structure recognition interface to realize efficient binding of aptamer and high-sensitivity analysis of target substances is the key to this technology.

[0003] Currently, functionalized nanoparticles are used as basic building blocks to modify aptamer probes on magnetic nanoparticles, mainly including sol-gel (sol-gel) method, biological method and nano gold bridging method, etc. In the sol-gel method, based on the sol-gel reaction of Fe3O4 and organosiloxane, amino groups are introduced onto the surface of magnetic beads, and then the carboxylated aptamer is bonded through reaction with the amino groups to realize the modification and bonding of aptamer. In the biological method, based on the specific reaction of streptavidin-biotin, the aptamer with biotin is bonded to the surface of streptavidin-coupled magnetic beads, and the indicator probe is released into the solution by the binding of target substances to the aptamer backbone, so as to determine the concentration of target substances by the change of signal intensity of the indicator probe. In the nano gold bridging method, nano gold is used to bridge the aptamer with thiol groups, such as preparing Fe3O4-PEI nanoparticles by co-precipitation method, then introducing amino groups on the surface of magnetic beads by coating polyethyleneimine PEI to directly adsorb gold particles, and bonding with aptamer with terminal thiol groups to realize the bonding of aptamer on the surface of magnetic core. However, these technologies have problems such as decrease of specific surface area after polymer modification of Fe3O4 particles, limited density of amino surface modification, low binding efficiency of nano gold, and disordered bonding of aptamer on nano gold.

[0004] Metal-organic framework (MOF) materials have ultra-high specific surface area, excellent chemical and thermal stability, and abundant active sites, etc. When combined with magnetic beads, they show good performance in micro-separation, enrichment and sensing analysis. Scientists have developed Fe3O4@MOF nanoparticle technology by in-situ growth of carboxylated magnetic beads and metal ions. Based on the reaction of amino groups introduced on the MOF ligand with the carboxylated aptamer, or the bonding of gold nanoparticles and the subsequent fixation of thiolated aptamer, the application of Fe3O4@MOF@aptamer biological probes has been developed. However, due to the limited number of amino groups introduced on the MOF ligand, the number of carboxylated aptamers and gold nanoparticles bonded in the subsequent reaction is limited. At the same time, the modification of aptamers on magnetic nanoparticles is difficult and requires high technical requirements, and the modification efficiency is low. Moreover, the active metal end groups on MOF are prone to adsorb aptamers, which can lead to the adsorption of aptamer chains on the surface of MOF and the occurrence of lodging phenomenon, thereby inhibiting the reaction activity of aptamers on the surface of MOF-gold composite material. Therefore, it is necessary to improve the content of MOF on the surface of magnetic nanoparticles, inhibit the activity of central ions of MOF, and passivate the activity of non-bonding sites of gold nanoparticles based on the self-assembly of MOF and metal passivation technology, to construct gold-magnetic composite materials with specific structures, and realize the ordered combination of aptamers on the surface of magnetic nanoparticles, and improve the reaction activity of aptamer magnetic nanoprobe. SUMMARY

[0005] The present application aims to provide a kind of based on multilayer organic metal framework-gold nanoparticle mediated aptamer functionalized magnetic nanoprobe and its preparation method.The prepared aptamer functionalized magnetic nanoprobe has the characteristics of rich amino group, high gold nanoparticle loading efficiency and ordered aptamer bonding, realizes the efficient assembly of aptamer probe, and can improve the efficient specific recognition separation and sensitive analysis of target.

[0006] To achieve the above-mentioned purpose, the technical scheme is as follows:

[0007] A kind of based on multilayer organic metal framework-gold nanoparticle mediated aptamer functionalized magnetic nanoprobe, which is coated with polydopamine (PDA) magnetic nanoparticles, and in-situ growth is formed on its multilayer organic metal framework-gold composite material, then the magnetic nanoparticles loaded with composite material are used as carrier to bond aptamer fluorescent probe, and thiol reagent is used for blocking, to form the aptamer functionalized magnetic nanoprobe;

[0008] The multilayer organic metal framework-gold composite material is specifically a multilayer structure organic metal framework and gold nanoparticle bonded by in-situ growth twice on the polydopamine coated magnetic nanoparticles and passivated by central metal ions;The aptamer fluorescent probe is formed by hybridizing the 5' end thiolated nucleic acid aptamer DNA chain with the 5' end fluorescent label group cDNA complementary chain.

[0009] Further, the twice in-situ growth is specifically to fix the zirconium (IV) metal center ion by using the amino group of polydopamine, and to perform the first in-situ growth of the organic metal framework material by using terephthalic acid as a ligand to form an amino UIO-66 (UIO1), and then to perform the second in-situ growth (UIO2) of the organic metal framework material by using the obtained amino UIO-66 as a crystal seed.

[0010] The center metal ion passivation is to perform a passivation reaction on the product after the twice in-situ growth by using N α ,N α - Bis (carboxymethyl) -L-lysine hydrate to form a lysine product.

[0011] Further, the 5' end of the aptamer DNA chain with a thiol group is an aptamer against Jatopin acid, and the structure sequence is 5'-SH-C6-CCACCAACGAGAGTCAGAAAACCATGGTGGG-3'; the structure sequence of the 5' end of the cDNA complementary chain with a fluorescent labeling group is 5'-FAM-GGTTTTCTGAC-3'.

[0012] Further, the thiol reagent is mercaptohexanol.

[0013] Further, the magnetic nanoparticles are spherical Fe3O4 particles.

[0014] The preparation method of the aptamer functionalized magnetic nanoprobe based on the multi-layer organic metal framework-nanogold mediation specifically includes the following steps:

[0015] (1) Preparation of polydopamine coated magnetic nanoparticles:

[0016] 8.1 g of ferric chloride hexahydrate was weighed and dissolved in 200 mL of ethylene glycol to prepare solution A; 12.0 g of anhydrous sodium acetate and 2.0 g of sodium citrate dihydrate were added to solution A, and stirred and dissolved to form a uniform and stable solution B; the obtained solution B was transferred to a polytetrafluoroethylene lined high-pressure reaction kettle, which was placed in a 200℃ oven, and reacted for 10 hours; after the reaction kettle was naturally cooled to room temperature, the precipitate was separated, washed with deionized water and ethanol three times alternately, and then vacuum dried at 60℃ for 24h to obtain Fe3O4 magnetic nanoparticle powder; 200mg of Fe3O4 magnetic nanoparticle powder, 240mg of dopamine hydrochloride and 240mg of tris-hydroxymethyl aminomethane were weighed and ultrasonically dispersed in 200mL of ultrapure water under ice water bath, and then stirred and reacted at room temperature for 24h; the reaction product was then washed with deionized water several times to obtain polydopamine coated magnetic nanoparticles Fe3O4@PDA;

[0017] (2) Preparation of lysine Fe3O4@PDA@UIO / UIO2 nanoparticles:

[0018] Fe3O4@PDA 100 mg, zirconium chloride 71.4 mg, 2-amino terephthalic acid 50.78 mg, acetone 70 mL and acetic acid 4.2 mL were weighed and uniformly mixed, and after ultrasonic treatment for 10 min, they were transferred into a polytetrafluoroethylene-lined high-pressure reaction kettle, and after reaction at 100°C for 24 h, they were washed and dried to obtain Fe3O4@PDA@UIO1; the prepared Fe3O4@PDA@UIO1 was readded into the reaction kettle, and after continuous reaction at the same growth concentration and reaction condition for 24 h, it was washed with water and ethanol alternately for 3 times to obtain Fe3O4@PDA@UIO1 / UIO2; 2 mg of Fe3O4@PDA@UIO1 / UIO2 was dispersed in 200 μL of 0.02 mol / L N α ,N α -lysine Fe3O4@PDA@UIO / UIO2 (Fe3O4@PDA@K-UIO1 / K-UIO2) nanoparticles were obtained by washing the product with double-distilled water after reaction in the solution of bis(carboxymethyl)-L-lysine hydrate at room temperature for 30 min;

[0019] (3) Preparation of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles:

[0020] 98 mL of double-distilled water and 2 mL of 50 mmol / L chloroauric acid solution were added into a 250 mL three-necked flask, and after uniform stirring, the three-necked flask was placed in an oil bath at 110°C and boiled for 5 min; after the liquid in the flask boiled, 10 mL of 38.8 mmol / L sodium citrate solution was rapidly added under magnetic stirring, and when the color of the reaction liquid changed from light yellow to wine red, boiling reflux was continued for 20 min, and then heating was stopped, and the liquid was cooled to room temperature, filtered with a 0.22 μm filter membrane to obtain a gold nanoparticle (AuNPs) solution; Fe3O4@PDA@K-UIO1 / K-UIO2 nanoparticles obtained in step (2) were added and left overnight, and after magnetic separation to remove the excess gold nanoparticle solution on the top, Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles were obtained;

[0021] (4) Preparation of aptamer fluorescent probe

[0022] Take the 5' end of the thiol group of the nucleic acid aptamer DNA chain and the 5' end of the fluorescent label group of the cDNA complementary chain freeze-dried reagent, centrifuge at room temperature at 10000 rpm for 5 min, then add ultrapure water to prepare an aptamer solution and a fluorescent complementary chain solution with a concentration of 100 mu M respectively; the two solutions are activated in a 90 DEG C constant temperature water bath for 10 min, and then quickly transferred to a 0 DEG C ice water mixture for cooling for 10 min to obtain an aptamer stock solution and a fluorescent complementary chain stock solution; the obtained aptamer stock solution, fluorescent complementary chain stock solution and PBS buffer salt solution (10 mmol / L PBS, pH=7.50, 500 mmol / L NaCl) are mixed and incubated at 55 DEG C for 2 h to obtain an aptamer fluorescent probe solution;

[0023] (5) Preparation of aptamer functionalized magnetic nanoprobes:

[0024] In 50 mu L of 10 mu mol / L aptamer fluorescent probe solution, 0.6 mg of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles prepared in step (3) is added, and after oscillation dispersion at 25 DEG C for 15 min, magnetic separation is carried out; then 40 mu L of 2 mol / L mercaptohexanol solution is removed and added to the dispersion liquid of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe, and after oscillation at 25 DEG C for 1 h, magnetic separation is carried out, to obtain mercaptohexanol blocked Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe.

[0025] Further, in step (4), the volume ratio of the aptamer stock solution, the fluorescent complementary chain stock solution and the PBS buffer salt solution used in the mixing is 1:1:8.

[0026] The obtained aptamer functionalized magnetic nanoprobes based on multilayer organic metal framework-nano gold mediation can be used for detecting halichondramide.

[0027] The present application utilizes polydopamine PDA to physically coat Fe3O4 magnetic nanoparticles, thereby introducing amino groups on the surface of Fe3O4, for realizing in-situ growth of organic metal framework UIO1 / UIO2, and introducing lysine on the surface of MOF to realize passivation of metal residues, forming MOF structure with high specific surface area, passivated central metal ions and rich amino groups; on this basis, the composite material is coupled with nano gold, bonded with aptamer fluorescent probe and introduced with thiol small molecules for passivation and shielding of nano gold blank sites, to avoid aptamer lodging phenomenon.

[0028] The present application has the following advantages:

[0029] The application realizes the in-situ growth of the organic metal framework material on the surface of Fe3O4 multiple times by directly chelating metal ions with polydopamine, forms the composite structure of UIO1 / UIO2 with high specific surface area, and introduces N by post modification α N α The double (carboxymethyl acid)-L-lysine hydrate realizes the further amination of the MOF and the passivation of the metal end point, thereby forming the Fe3O4@MOF core-shell functional material with high specific surface area, passivated metal ions and rich amino groups; on this basis, the nano-gold is efficiently coupled with the rich amino groups of the MOF, bonded with the thiol aptamer fluorescent probe, and further passivated by introducing the thiol auxiliary reagent, so as to realize the saturated coverage of the nano-gold surface and the morphology regulation of the aptamer, and the magnetic nanoparticle with high-density aptamer fluorescent probe loaded on the surface is prepared, which avoids the problems such as low binding amount of nano-gold on the MOF surface, agglomeration, lodging and disordered winding of the aptamer during the assembly on the MOF surface, and realizes the efficient loading and ordered assembly of the aptamer probe on the magnetic nano material.

[0030] The aptamer functionalized magnetic nano probe prepared by the application is applied to the detection of okadaic acid (OA), has good detection specificity, keeps the fluorescence intensity of 1 ng / mL OA above 120000 (A.U.), and can effectively avoid the interference of other toxins. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a process schematic diagram for preparing the aptamer functionalized magnetic nano probe of the application.

[0032] Figure 2 It is a spectrum for detecting okadaic acid (OA) by using the aptamer functionalized magnetic nano probe prepared in Example 1.

[0033] Figure 3 It is a linear working curve obtained by detecting okadaic acid (OA) by using the aptamer functionalized magnetic nano probe prepared in Example 1.

[0034] Figure 4 It is a result graph of anti-interference performance test of the aptamer functionalized magnetic nano probe prepared in Example 1. DETAILED DESCRIPTION

[0035] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described below in combination with specific embodiments, but the application is not limited to this.

[0036] Example 1

[0037] A preparation method of an aptamer functionalized magnetic nano probe based on multi-layer organic metal framework-nano gold mediation, the specific steps are as follows:

[0038] (1) Preparation of polydopamine-coated magnetic nanoparticles:

[0039] Weigh 8.1 g of ferric chloride hexahydrate into 200 mL of ethylene glycol to prepare solution A; add 12.0 g of anhydrous sodium acetate and 2.0 g of sodium citrate dihydrate into solution A, stir and dissolve to form a uniform and stable solution B; transfer the obtained solution B into a polytetrafluoroethylene-lined high-pressure reaction kettle, place it in a 200°C oven, and react for 10 hours; after the reaction kettle is naturally cooled to room temperature, separate the precipitate, wash it with distilled water and ethanol three times alternately, and then vacuum dry it at 60°C for 24 h to obtain Fe3O4 magnetic nanoparticle powder; weigh 200 mg of Fe3O4 magnetic nanoparticle powder, 240 mg of dopamine hydrochloride PDA, and 240 mg of tris(hydroxymethyl)aminomethane, and ultrasonically disperse them in 200 mL of ultrapure water under ice water bath, stir at room temperature for 24 h, and then wash the post-reaction product with distilled water several times to obtain polydopamine-coated Fe3O4 magnetic nanoparticles Fe3O4@PDA.

[0040] (2) Preparation of lysine-modified Fe3O4@PDA@UIO1 / UIO2 nanoparticles:

[0041] Weigh 100 mg of Fe3O4 nanoparticles, 71.4 mg of zirconium chloride, 50.78 mg of 2-aminoterephthalic acid, 70 mL of acetone, and 4.2 mL of acetic acid, mix them thoroughly and uniformly, ultrasonically treat them for 10 min, then transfer them into a polytetrafluoroethylene-lined high-pressure reaction kettle, react at 100°C for 24 h, wash and dry them to obtain Fe3O4@PDA@UIO1; add 100 mg of the prepared Fe3O4@PDA@UIO1 into the above reaction kettle, and also add 71.4 mg of zirconium chloride, 50.78 mg of 2-aminoterephthalic acid, 70 mL of acetone, and 4.2 mL of acetic acid, mix them thoroughly and uniformly, ultrasonically treat them for 10 min, then react at 100°C for 24 h; wash the obtained product Fe3O4@PDA@UIO / UIO2 with water and ethanol alternately three times; then weigh 2 mg of the obtained Fe3O4@PDA@UIO / UIO2, disperse it in 200 μL of 0.02 mol / L N α N α - Bis(carboxymethyl)-L-lysine hydrate, react at room temperature for 30 min, wash the product with distilled water to obtain Fe3O4@PDA@K-UIO1 / K-UIO2 nanoparticles.

[0042] (3) Preparation of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles:

[0043] In a 250 mL three-necked flask, 98 mL of double-distilled water and 2 mL of 50 mmol / L chloroauric acid solution were added, and after being stirred well, the three-necked flask was placed in an oil bath at 110°C and boiled for 5 min under continuous magnetic stirring. After the liquid in the flask boiled, 10 mL of 38.8 mmol / L sodium citrate solution was quickly added, and when the color of the reaction liquid changed from light yellow to wine red, boiling reflux was continued for 20 min, and then heating was stopped. After cooling to room temperature, the solution was filtered with a 0.22 μm filter membrane to obtain a gold nanoparticle (AuNPs) solution with a particle size of 20-30 nm. The Fe3O4@PDA@K-UIO1 / K-UIO2 nanoparticles obtained in step (2) were added to the above gold nanoparticle solution and left overnight. After magnetic separation, the excess gold nanoparticle solution was removed to obtain Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles.

[0044] (4) Preparation of aptamer fluorescent probe

[0045] The 5'-thiol-containing aptamer DNA chain (sequence: 5'-SH-C6-CCACCAACGAGAGTCAGAAAACCATGGTGGG-3') and the freeze-dried reagent of the modified fluorescein complementary short cDNA (5'-FAM-GGTTTTCTGAC-3') were placed in a centrifuge at room temperature at a speed of 10,000 rpm for 5 min, and then ultrapure water was added to prepare an aptamer and fluorescent complementary chain solution with a concentration of 100 μM. The two solutions were activated in a 90°C constant temperature water bath for 10 min, then quickly transferred to an ice-water mixture at 0°C for cooling for 10 min to obtain the aptamer and fluorescent complementary chain stock solution. A PBS buffer solution (10 mmol / L PBS, pH=7.50, 500 mmol / L NaCl) was prepared, and the three were mixed according to the ratio of aptamer: complementary chain: PBS buffer = 1:1:8 (v / v / v), and incubated at 55°C for 2 h to obtain the aptamer fluorescent probe solution.

[0046] (5) Preparation of aptamer functionalized magnetic nanoprobe:

[0047] In 50 μL of the aptamer fluorescent probe solution prepared in step (4) with a concentration of 10 μmol / L, 0.6 mg of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles prepared in step (3) was added, and placed in a constant temperature mixing instrument for oscillation dispersion at 25°C for 15 min, and then subjected to magnetic separation. Then, 40 μL of 2 mol / L mercaptohexanol solution was added to the dispersion of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe, and subjected to magnetic separation after oscillation at 25°C in the constant temperature mixing instrument for 1 h, to obtain mercaptohexanol-closed Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe.

[0048] Dynamic light scattering (DLS) was performed on the prepared Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs, Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe and mercaptohexanol-closed Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe, and the results showed that the molecular particle sizes thereof were 261 nm, 266 nm and 273 nm, respectively, which was close to the theoretical value 271.2 nm of the upright distribution of aptamer DNA chain, proving that the upright distribution state of the aptamer on the MOF surface was obviously improved.

[0049] Example 2

[0050] The Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe prepared in Example 1 was used to detect okadaic acid (OA), specifically, 0.60 mg of the Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe was taken, 40 μL of okadaic acid OA solution with different concentrations was added, and the sample was incubated at room temperature for 120 min, and then the supernatant was collected by magnetic separation, and the fluorescence signal of the sample was detected. The fluorescence measurement conditions were as follows: the laser-induced fluorescence (LIF) excitation wavelength was 497 nm, the emission wavelength was 512 nm, the quartz capillary specification was 100 μm×500 mm, the mobile phase was PBS buffer solution with pH=7.5, the flow rate was 0.2 mL / min, the sample volume was 0.06-0.1 μL, and the PBS buffer solution was composed of 10 mmol / L NaH2PO4, 10 mmol / L Na2HPO4 and 500 mmol / L NaCl. The results are shown in Figure 2 3

[0051] Figure 2 3 ​​​​As can be seen, under the above measurement conditions, the fluorescence response intensity of the magnetic nanoprobe sensing system increases with increasing OA concentration, and it exhibits a good linear range (R0). 2 = 0.997), and the detection limit was calculated to be 15 ng / L using the 3-times standard deviation method.

[0052] Under the above testing conditions, the Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe prepared in Example 1 was used to detect 1 ng / mL of okata scleroderma and 20 ng / mL of tetrodotoxin, tetrodotoxin, and domoic acid, respectively. The results are shown in [Figure 1]. Figure 4 .

[0053] Depend on Figure 4 It is evident that the aptamer magnetic nanoprobe mediated by multilayer organometallic framework-gold nanoparticles exhibits excellent detection specificity, maintaining a fluorescence intensity above 120,000 (AU) for 1 ng / mL of OA, while the fluorescence intensity of other toxins at concentrations 20 times that of OA is only 1,000–2,000 (AU). This demonstrates that the fluorescence sensing system based on multilayer organometallic framework-gold nanoparticles has good anti-interference properties.

[0054] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A multi-layered organic metal framework-nanogold mediated aptamer functionalized magnetic nanoprobe, characterized in that: The polydopamine-coated magnetic nanoparticles are coated with a multi-layer organic metal framework-nano gold composite material, and then a ligand fluorescent probe is bonded to the magnetic nanoparticles loaded with the composite material, and a thiol reagent is used for blocking to form the aptamer functionalized magnetic nano probe; The multi-layer organic metal framework-nano gold composite material is a multi-layer structure organic metal framework and nano gold bonded by twice in-situ growth and central metal ion passivation on the polydopamine-coated magnetic nanoparticles; the aptamer fluorescent probe is formed by hybridization of a 5'-end thiol group-containing nucleic acid aptamer DNA chain and a 5'-end fluorescent label group-containing cDNA complementary chain; The twice in-situ growth is to fix zirconium (IV) metal center ions by using the amino group of polydopamine, and to perform one-time in-situ growth of the organic metal framework material by using terephthalic acid as a ligand to form an amino UIO-66, and to perform twice in-situ growth of the organic metal framework material by using the obtained amino UIO-66 as a seed crystal; The center metal ion passivation is passivation of the twice in-situ grown product with N α ,N α -bis(carboxymethyl)-L-lysine hydrate to form a lysinated product. 2.The multi-layer organic metal framework-nanogold mediated aptamer functionalized magnetic nanoprobe based on claim 1, wherein: The 5'-end thiol group-containing aptamer DNA chain is an anti-leucettidine acid aptamer, and its structure sequence is 5'-SH-C6-CCACCAACGAGAGTCAGAAAACCATGGTGGG-3'; the structure sequence of the 5'-end fluorescent label group-containing cDNA complementary chain is 5'-FAM-GGTTTTCTGAC-3'. 3.The multi-layer organic metal framework-nanogold mediated aptamer functionalized magnetic nanoprobe based on claim 1, characterized in that: The thiol reagent is thiol hexanol.

4. The method for preparing the multi-layer organic metal framework-nanogold mediated aptamer functionalized magnetic nanoprobe according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of polydopamine-coated magnetic nanoparticles: 8.1 g of iron trichloride hexahydrate is dissolved in 200 mL of ethylene glycol to prepare solution A; 12.0 g of anhydrous sodium acetate and 2.0 g of sodium citrate dihydrate are added to solution A, and stirred and dissolved to form a uniform and stable solution B; the obtained solution B is transferred into a polytetrafluoroethylene-lined high-pressure reaction kettle, which is placed in a 200°C oven, and reacted for 10 hours; After the reaction kettle is naturally cooled to room temperature, the precipitate is separated, washed with deionized water and ethanol three times alternately, and then vacuum dried at 60°C for 24 h to obtain Fe3O4 magnetic nanoparticle powder; 200 mg of Fe3O4 magnetic nanoparticle powder, 240 mg of dopamine hydrochloride, and 240 mg of tris-hydroxymethyl aminomethane are ultrasonically dispersed in 200 mL of ultrapure water under ice water bath, and stirred and reacted at room temperature for 24 h, and then the reaction product is washed with deionized water several times to obtain polydopamine-coated magnetic nanoparticles Fe3O4@PDA; (2) Preparation of lysine Fe3O4@PDA@UIO / UIO2 nanoparticles: Fe3O4@PDA 100 mg, zirconium chloride 71.4 mg, 2-amino terephthalic acid 50.78 mg, acetone 70 mL and acetic acid 4.2 mL were weighed and mixed uniformly, and then transferred into a polytetrafluoroethylene-lined high-pressure reaction kettle after ultrasonic treatment for 10 min. After reaction at 100°C for 24 h, Fe3O4@PDA@UIO1 was obtained after washing and drying. The prepared Fe3O4@PDA@UIO1 was readded into the reaction kettle, and continued to react for 24 h under the same growth concentration and reaction conditions. After washing with water and ethanol alternately for 3 times, Fe3O4@PDA@UIO1 / UIO2 was obtained. 2 mg of Fe3O4@PDA@UIO1 / UIO2 was dispersed in 200 μL of 0.02 mol / L N α ,N α -After reaction at room temperature for 30 min, the product was washed with double distilled water to obtain Fe3O4@PDA@K-UIO1 / K-UIO2 nanoparticles. (3) Preparation of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles: In a 250 mL three-necked flask, 98 mL of double-distilled water and 2 mL of 50 mmol / L chloroauric acid solution were added, and the three-necked flask was placed in an oil bath at 110°C and boiled for 5 min after being stirred evenly. After the liquid in the flask boiled, 10 mL of 38.8 mmol / L sodium citrate solution was quickly added under magnetic stirring, and the reaction liquid was boiled and refluxed for 20 min after the color changed from light yellow to wine red. Then the heating was stopped, and the liquid was cooled to room temperature. The AuNPs solution was obtained by filtration with a 0.22 μm filter membrane. Then the Fe3O4@PDA@K-UIO1 / K-UIO2 nanoparticles obtained in step (2) were added and placed overnight. After removing the excess gold nanoparticles solution by magnetic separation, the Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles were obtained. (4) Preparation of aptamer fluorescent probe The 5' end thiol-containing nucleic acid aptamer DNA chain and the 5' end thiol-containing nucleic acid aptamer DNA chain were freeze-dried and centrifuged at 10000 rpm for 5 min at room temperature. Then, ultrapure water was added to prepare aptamer solution and fluorescent complementary chain solution with a concentration of 100 μM. The two solutions were activated in a 90°C constant temperature water bath for 10 min, and then quickly transferred to an ice-water mixture at 0°C for cooling for 10 min to obtain aptamer stock solution and fluorescent complementary chain stock solution. The obtained aptamer stock solution, fluorescent complementary chain stock solution and 10 mmol / L PBS buffer salt solution were mixed and incubated at 55°C for 2 h to obtain the aptamer fluorescent probe solution. (5) Preparation of aptamer functionalized magnetic nanoprobe: In 50 μL of 10 μmol / L aptamer fluorescent probe solution, 0.6 mg of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs nanoparticles prepared in step (3) was added, and the mixture was dispersed by shaking at 25°C for 15 min, and then subjected to magnetic separation. Then, 40 μL of 2 mol / L mercaptohexanol solution was added to the dispersion of Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe, and the mixture was shaken at 25°C for 1 h, and then subjected to magnetic separation to obtain mercaptohexanol-capped Fe3O4@PDA@K-UIO1 / K-UIO2@AuNPs@aptamer probe.

5. The method for preparing multi-layer organic metal framework-nanogold mediated aptamer functionalized magnetic nanoprobe according to claim 4, characterized in that: In step (4), the volume ratio of aptamer stock solution, fluorescent complementary chain stock solution and PBS buffer salt solution was 1:1:

8.

6. A multi-layer organic metal framework-nanogold-mediated aptamer functionalized magnetic nanoprobe based on claim 1 for detecting leucettine.

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