CN116482067A - Aptamer functionalized magnetic nano probe based on multilayer organic metal framework-nano Jin Jie guide and preparation method thereof - Google Patents
Aptamer functionalized magnetic nano probe based on multilayer organic metal framework-nano Jin Jie guide and preparation method thereof Download PDFInfo
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 92
- 239000000523 sample Substances 0.000 title claims abstract description 43
- 229910052751 metal Inorganic materials 0.000 title claims abstract description 35
- 239000002184 metal Substances 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 229920001690 polydopamine Polymers 0.000 claims abstract description 62
- 239000010931 gold Substances 0.000 claims abstract description 33
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 30
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- 230000000295 complement effect Effects 0.000 claims abstract description 19
- 238000011065 in-situ storage Methods 0.000 claims abstract description 16
- 125000003396 thiol group Chemical group [H]S* 0.000 claims abstract description 16
- 239000002131 composite material Substances 0.000 claims abstract description 13
- 239000003446 ligand Substances 0.000 claims abstract description 10
- 108091008104 nucleic acid aptamers Proteins 0.000 claims abstract description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 9
- 239000002299 complementary DNA Substances 0.000 claims abstract description 8
- 239000013207 UiO-66 Substances 0.000 claims abstract description 5
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- GPNNOCMCNFXRAO-UHFFFAOYSA-N 2-aminoterephthalic acid Chemical compound NC1=CC(C(O)=O)=CC=C1C(O)=O GPNNOCMCNFXRAO-UHFFFAOYSA-N 0.000 claims description 4
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 238000001215 fluorescent labelling Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- DUNKXUFBGCUVQW-UHFFFAOYSA-J zirconium tetrachloride Chemical compound Cl[Zr](Cl)(Cl)Cl DUNKXUFBGCUVQW-UHFFFAOYSA-J 0.000 claims description 4
- CTENFNNZBMHDDG-UHFFFAOYSA-N Dopamine hydrochloride Chemical compound Cl.NCCC1=CC=C(O)C(O)=C1 CTENFNNZBMHDDG-UHFFFAOYSA-N 0.000 claims description 3
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- 239000002244 precipitate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- 229960000999 sodium citrate dihydrate Drugs 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
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- GBNDTYKAOXLLID-UHFFFAOYSA-N zirconium(4+) ion Chemical compound [Zr+4] GBNDTYKAOXLLID-UHFFFAOYSA-N 0.000 claims description 2
- 125000002524 organometallic group Chemical group 0.000 claims 4
- 230000008901 benefit Effects 0.000 abstract description 3
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- 239000004472 Lysine Substances 0.000 abstract description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract description 2
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
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- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
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- RPQXVSUAYFXFJA-UHFFFAOYSA-N saxitoxin hydrate Natural products NC(=O)OCC1N=C(N)N2CCC(O)(O)C22NC(N)=NC12 RPQXVSUAYFXFJA-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an aptamer functionalized magnetic nano-probe based on multilayer organic metal framework-nano Jin Jie guide and a preparation method thereof, wherein the aptamer functionalized magnetic nano-probe is formed by taking magnetic nano-particles which are coated by polydopamine and are provided with multilayer organic metal framework-nano-gold composite materials in situ grown on the magnetic nano-particles as carriers, adopting a thiol reagent for sealing, and bonding a proper ligand fluorescent probe with high efficiency; the aptamer fluorescent probe is formed by hybridization of a nucleic acid aptamer DNA chain with a sulfhydryl group at the 5 'end and a cDNA complementary chain with a fluorescent marking group at the 5' end; the multi-layer organic metal framework-nano gold composite material is formed by bonding lysine amino UIO-66 with a multi-layer structure and nano gold. The aptamer functionalized magnetic nano probe prepared by the invention has rich amino on the surface, can be efficiently bonded with nano gold and bridged with an aptamer fluorescent probe, and has the advantages of high binding efficiency with a target substance, strong recognition specificity and the like.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, and provides an aptamer magnetic nano probe based on a multilayer organic metal framework-nano Jin Jie guide and a preparation method thereof.
Background
The field halichondrin is a common red tide algae toxin, is enriched in digestive glands of various marine mollusks, and can cause diarrhea poisoning after eating marine products polluted by the field halichondrin. Therefore, developing field halichondrin is significant for guaranteeing food safety and consumer health. Currently, the monitoring technology of field halichondrin mainly comprises chromatography, sensor method, biological method and the like. Among the sensing analysis techniques, the analysis technique using a nucleic acid aptamer as a recognition element has been widely paid attention to. The nucleic acid aptamer has the advantages of good affinity, easy separation, stable signal and the like, and the functionalized magnetic nano probe prepared by coupling the aptamer with the magnetic nano particles is used as an emerging targeting nano probe, so that the nucleic acid aptamer has rapid application and development in selective extraction, separation and detection of red tide algae toxins. In the research of the aptamer nano probe recognition technology, how to construct a stable recognition interface with a specific structure so as to realize efficient binding of the aptamer and high-sensitivity analysis of a target object is a key for realizing the technology.
Currently, the technology of modifying an aptamer probe with a magnetic nanoparticle mainly includes a sol-gel (sol-gel) method, a biological method, a nano Jin Qiaolian method, and the like by using a functionalized nanoparticle as a basic building unit. In the sol-gel process, fe-based 3 O 4 And (3) carrying out sol-gel reaction with organosiloxane, introducing amino groups on the surfaces of the magnetic beads, and carrying out reaction with carboxylated aptamer to realize aptamer modification and bonding. In biological methods, chains are basedThe specific reaction of the streptavidin-biotin bonds the aptamer connected with the biotin to the surface of the streptavidin-coupled magnetic bead, and the target substance is combined with the aptamer main chain to release the indication probe into the solution, so that the concentration of the target substance is determined through the change of the signal intensity of the indication probe. In the nano-gold bridging method, nano-gold is used for bridging an aptamer with a sulfhydryl group, such as the preparation of Fe by a coprecipitation method 3 O 4 And (3) directly adsorbing gold particles by coating polyethyleneimine PEI on the surface of the magnetic beads to introduce amino groups, and bonding with an aptamer with a terminal sulfhydryl group to realize bonding of the proper ligand on the surface of the magnetic core. However, polymer modified Fe exists in these techniques 3 O 4 The specific surface after particles are reduced, the amino surface modification density is limited, the nano gold binding efficiency is low, and the aptamer is disordered in nano gold binding.
The metal organic framework MOF material has ultrahigh specific surface area, excellent chemical and thermal stability, rich active sites and the like, and combines the metal organic framework MOF material with magnetic beads, thereby exhibiting good performance in the fields of micro-differential enrichment and sensing analysis. Scientists developed Fe by in situ growth of carboxylated magnetic beads and metal ions 3 O 4 @mof nanoparticle technology. Based on the reaction of amino groups introduced on MOF ligand and carboxyl aptamer or the binding of nano gold to re-fix sulfhydryl aptamer, fe 3 O 4 The use of @ MOF @ ligand bioprobes has been developed. However, due to the limited amino introduced on the MOF ligand, the quantity of the carboxyl aptamer and the nano gold bonded in the subsequent reaction is limited, meanwhile, the technical requirements on the modification difficulty of the aptamer on the magnetic nanoparticle are high, the modification efficiency is low, and the active metal end group on the MOF is easy to adsorb the aptamer, so that the aptamer chain is easy to adsorb on the surface of the MOF and lodging occurs, and the reactivity of the aptamer on the surface of the MOF-nano gold composite material is inhibited. Therefore, it is necessary to construct gold-magnetic composite materials with specific structures based on MOF self-assembly and metal passivation technology, so as to improve the MOF content of the magnetic nanoparticle surface, inhibit the activity of MOF center ions and passivate the activity of non-binding sites of nano gold, realize the orderly binding of the aptamer on the magnetic nanoparticle surface and improve the aptamer magnetic nanoprobeReactivity of the needle.
Disclosure of Invention
The invention aims to provide an aptamer functionalized magnetic nano probe based on a multilayer organic metal framework-nano Jin Jie guide and a preparation method thereof. The prepared aptamer functionalized magnetic nano probe has the characteristics of rich amino, high nano gold loading efficiency, ordered aptamer bonding and the like, realizes efficient assembly of the aptamer probe, and can improve efficient specific identification separation and sensitive analysis of a target object.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an aptamer functionalized magnetic nano probe based on a multilayer organic metal framework-nano Jin Jie guide is characterized in that Polydopamine (PDA) is utilized to coat magnetic nano particles, a multilayer organic metal framework-nano gold composite material is formed on the magnetic nano particles through in-situ growth, then the magnetic nano particles loaded with the composite material are used as carrier bonds to be suitable ligand fluorescent probes, and a sulfhydryl reagent is adopted for sealing, so that the aptamer functionalized magnetic nano probe is formed;
the multi-layer organic metal frame-nano gold composite material is formed by bonding a multi-layer structure organic metal frame formed by two in-situ growth and central metal ion passivation on polydopamine coated magnetic nano particles with nano gold; the aptamer fluorescent probe is formed by hybridization of a nucleic acid aptamer DNA chain with a sulfhydryl group at the 5 'end and a cDNA complementary chain with a fluorescent marking group at the 5' end.
Further, the two in-situ growth is specifically to fix zirconium (IV) metal center ions by utilizing amino groups of polydopamine, perform primary in-situ growth of the organic metal framework material by taking terephthalic acid as a ligand to form amino UIO-66 (UIO 1), and perform secondary in-situ growth (UIO 2) of the organic metal framework material by taking the obtained amino UIO-66 as a seed crystal;
the passivation of the central metal ion is to make the product after two in-situ growth and N α ,N α -passivating the bis (carboxymethyl) -L-lysine hydrate to form a lysinated product.
Further, the saidThe aptamer DNA chain with mercapto at 5 'end is aptamer of anti-field halichondrin, its structural sequence is 5' -SH-C 6 CCACCAACGAGAGTCAGAAAACCATGGTGGG-3'; the structural sequence of the cDNA complementary strand with the fluorescent labeling group at the 5' end is 5' -FAM-GGTTTTCTGAC-3'.
Further, the sulfhydryl reagent is sulfhydryl hexanol.
Further, the magnetic nano particles are spherical Fe 3 O 4 And (3) particles.
The preparation method of the aptamer functionalized magnetic nano probe based on multilayer organic metal framework-nano Jin Jie guide specifically comprises the following steps:
(1) Preparation of polydopamine coated magnetic nanoparticles:
8.1g of ferric trichloride hexahydrate is weighed and dissolved in 200mL of ethylene glycol to prepare a solution A; adding 12.0g of anhydrous sodium acetate and 2.0g of sodium citrate dihydrate into the solution A, and stirring and dissolving until a uniform and stable solution B is formed; transferring the obtained solution B into a high-pressure reaction kettle with a polytetrafluoroethylene lining, and placing the high-pressure reaction kettle into a baking oven with the temperature of 200 ℃ for reaction for 10 hours; after the reaction kettle is naturally cooled to room temperature, separating out precipitate, alternately cleaning with secondary water and ethanol for three times, and vacuum drying at 60 ℃ for 24 hours to obtain Fe 3 O 4 Magnetic nanoparticle powder; weighing Fe 3 O 4 200mg of magnetic nanoparticle powder, 240mg of dopamine hydrochloride and 240mg of tris (hydroxymethyl) aminomethane, dispersing in 200mL of ultrapure water under ice water bath in an ultrasonic manner, stirring at room temperature for reaction for 24 hours, and washing the reaction product with secondary water for multiple times to obtain polydopamine coated magnetic nanoparticle Fe 3 O 4 @PDA；
(2) Lysinized Fe 3 O 4 Preparation of @ PDA @ UIO/UIO2 nanoparticles:
weighing Fe 3 O 4 PDA 100 mg, zirconium chloride 71.4 mg, 2-amino terephthalic acid 50.78 mg, acetone 70 mL and acetic acid 4.2 mL, after being fully and uniformly mixed, ultrasonic treated for 10min, transferred into a polytetrafluoroethylene lining high-pressure reaction kettle, reacted for 24h at 100 ℃, washed and dried to obtain Fe 3 O 4 Pda@uio1; the prepared Fe 3 O 4 Adding the @ PDA @ UIO1 into the reaction kettle again, continuously reacting for 24 hours under the same growth concentration and reaction condition, and alternately cleaning with water and ethanol for 3 times to obtain Fe 3 O 4 @PDA@UIO1/UIO2; weigh 2 mg Fe 3 O 4 @PDA@UIO1/UIO2, dispersed in 200. Mu.L of 0.02 mol/L N α ,N α After reaction of the bis (carboxymethyl) -L-lysine hydrate solution at room temperature for 30min, the product was washed with secondary water to give lysinated Fe 3 O 4 @PDA@UIO/UIO2（Fe 3 O 4 @PDA@K-UIO1/K-UIO 2) nanoparticles;
（3）Fe 3 O 4 preparation of @ PDA @ K-UIO1/K-UIO2@ AuNPs nanoparticles:
98mL of secondary water and 2mL of 50mmol/L chloroauric acid solution are added into a 250mL three-necked flask, and after the mixture is fully stirred, the three-necked flask is placed into an oil bath pot at 110 ℃ to be boiled for 5min; after the liquid in the bottle is boiled, 10mL of 38.8mmoL/L sodium citrate solution is rapidly added under the magnetic stirring condition, after the color of the reaction liquid is changed from light yellow to wine red, the reaction liquid is continuously boiled and refluxed for 20 minutes, then heating is stopped, stirring and cooling are carried out to room temperature, and a filter membrane with the size of 0.22 mu m is adopted for filtering, so that a nano gold (AuNPs) solution is obtained; adding Fe obtained in the step (2) 3 O 4 The @ PDA @ K-UIO1/K-UIO2 nano particles are placed overnight, and after the excessive nano gold solution on the upper layer is removed by magnetic separation, fe is obtained 3 O 4 The nano particles of @ PDA @ K-UIO1/K-UIO2@ AuNPs;
(4) Preparation of aptamer fluorescent probes
Taking a nucleic acid aptamer DNA strand with a sulfhydryl group at the 5 'end and a cDNA complementary strand freeze-drying reagent with a fluorescent labeling group at the 5' end, centrifuging at a speed of 10000 rpm for 5min at room temperature, and then adding ultrapure water to prepare an aptamer solution and a fluorescent complementary strand solution with a concentration of 100 mu M respectively; activating the two solutions in a constant-temperature water bath at 90 ℃ for 10min, and rapidly transferring the two solutions into an ice-water mixture at 0 ℃ for cooling for 10min to obtain an aptamer stock solution and a fluorescence complementary strand stock solution; uniformly mixing the obtained aptamer stock solution, the fluorescence complementary strand stock solution and PBS buffer salt solution (10 mmol/L PBS, pH=7.50, 500 mmol/L NaCl), and incubating at 55 ℃ for 2 h to obtain an aptamer fluorescent probe solution;
(5) Preparation of an aptamer functionalized magnetic nano probe:
adding 0.6mg of Fe prepared in the step (3) into 50 mu L of 10 mu mol/L aptamer fluorescent probe solution 3 O 4 The nano particles @ PDA @ K-UIO1/K-UIO2@ AuNPs are subjected to vibration dispersion for 15min at 25 ℃ and then subjected to magnetic separation; then 40. Mu.L of 2mol/L of mercaptohexanol solution was removed and added to Fe 3 O 4 In dispersion liquid of @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe, vibrating for 1h at 25 ℃ and then performing magnetic separation to obtain mercapto hexanol-blocked Fe 3 O 4 The probe is a @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe.
Further, in the mixing of the step (4), the volume ratio of the aptamer stock solution, the fluorescence complementary strand stock solution and the PBS buffer salt solution is 1:1:8.
The obtained aptamer functionalized magnetic nano probe based on multilayer organic metal framework-nano Jin Jie guide can be used for detecting halichondrin.
The invention physically coats Fe by utilizing polydopamine PDA 3 O 4 Magnetic nanoparticles, thus in Fe 3 O 4 Amino is introduced into the surface for realizing in-situ growth of the organic metal framework UIO1/UIO2, lysine is introduced into the surface of the MOF for realizing passivation of metal residues, and an MOF structure with high specific surface area, passivation of central metal ions and rich amino groups is formed; on the basis, the nano gold is coupled with nano gold to form a composite material, the composite material is bonded with an aptamer fluorescent probe, and a sulfhydryl small molecule passivation shielding nano gold blank site is introduced, so that the aptamer lodging phenomenon is avoided.
The invention has the remarkable advantages that:
the invention utilizes polydopamine to directly chelate metal ions to realize Fe 3 O 4 The organic metal frame material on the surface grows in situ for many times to form a composite structure with high specific surface of UIO1/UIO2, and N is introduced through post-modification α N α The bis (carboxyhc acid) -L-lysine hydrate achieves further amination of MOF and passivation of metal end points, thereby forming Fe with high specific surface area, metal ion passivation and rich amino group 3 O 4 @MOFA core-shell functional material; on the basis, through the efficient coupling of rich amino groups of MOF and nano gold, bonding with a sulfhydryl aptamer fluorescent probe, introducing sulfhydryl auxiliary reagent to further passivate the nano gold, the saturated coverage of the surface of the nano gold and the morphology regulation and control of the aptamer are realized, the magnetic nano particles of the surface high-density loaded aptamer fluorescent probe are prepared, the problems of low bonding amount of the nano gold on the surface of the MOF, aggregation, lodging, disordered winding and the like of the aptamer during the assembly of the surface of the MOF are avoided, and the efficient loading and ordered assembly of the nucleic acid aptamer probe on the magnetic nano material are realized.
The aptamer functionalized magnetic nano probe prepared by the invention is applied to detection of field halichondrin (OA), has good detection specificity, keeps fluorescence intensity of 1 ng/mL OA above 120000 (A.U.), and can effectively avoid interference of other toxins.
Drawings
FIG. 1 is a schematic process diagram of the preparation of an aptamer functionalized magnetic nanoprobe according to the present invention.
FIG. 2 is a graph showing detection of okadaic acid OA using the aptamer functionalized magnetic nano-probe prepared in example 1.
FIG. 3 is a linear working curve obtained by detecting okadaic acid OA using the aptamer functionalized magnetic nanoprobe prepared in example 1.
FIG. 4 is a graph showing the results of the anti-interference performance test of the aptamer functionalized magnetic nanoprobe prepared in example 1.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
The preparation method of the aptamer functionalized magnetic nano probe based on multilayer organic metal framework-nano Jin Jie guide comprises the following specific steps:
(1) Preparation of polydopamine coated magnetic nanoparticles:
8.1g of ferric trichloride hexahydrate was weighed into 200mL of ethylene glycol,preparing a solution A; adding 12.0g of anhydrous sodium acetate and 2.0g of sodium citrate dihydrate into the solution A, and stirring and dissolving until a uniform and stable solution B is formed; transferring the obtained solution B into a high-pressure reaction kettle with a polytetrafluoroethylene lining, and placing the high-pressure reaction kettle into a baking oven with the temperature of 200 ℃ for reaction for 10 hours; after the reaction kettle is naturally cooled to room temperature, separating out precipitate, alternately cleaning with secondary water and ethanol for three times, and vacuum drying at 60 ℃ for 24 hours to obtain Fe 3 O 4 Magnetic nanoparticle powder; weighing Fe 3 O 4 200mg of magnetic nanoparticle powder, 240mg of dopamine hydrochloride PDA and 240mg of tris (hydroxymethyl) aminomethane, dispersing in 200mL of ultrapure water under ice water bath in an ultrasonic manner, stirring for 24 hours at room temperature, and washing the reaction product with secondary water for multiple times to obtain polydopamine-coated Fe 3 O 4 Magnetic nanoparticle Fe 3 O 4 @PDA。
(2) Lysinized Fe 3 O 4 Preparation of @ PDA @ UIO1/UIO2 nanoparticles:
weighing Fe 3 O 4 Nanoparticle 100 mg, zirconium chloride 71.4 mg, 2-amino terephthalic acid 50.78 mg, acetone 70 mL and acetic acid 4.2 mL, mixing completely and uniformly, ultrasonic treating for 10min, transferring into polytetrafluoroethylene lining high pressure reactor, reacting at 100deg.C for 24 hr, washing, and drying to obtain Fe 3 O 4 Pda@uio1; the prepared Fe 3 O 4 Adding @ PDA @ UIO1 100 mg into the reaction kettle, adding 71.4 mg zirconium chloride, 50.78 mg of 2-amino terephthalic acid, 70 mL of acetone and 4.2 mL of acetic acid, mixing thoroughly and uniformly, performing ultrasonic treatment for 10min, reacting at 100deg.C for 24h, and collecting Fe 3 O 4 Alternately cleaning the @ PDA @ UIO/UIO2 with water and ethanol for 3 times; weighing the Fe obtained by 2 mg again 3 O 4 @PDA@UIO/UIO2, which was dispersed in 200. Mu.L of 0.02 mol/L N α N α In the (carboxymethyl) -L-lysine hydrate, the reaction is carried out for 30min at room temperature, and the product is washed by secondary water to obtain Fe 3 O 4 @PDA@K-UIO1/K-UIO2 nanoparticles.
（3）Fe 3 O 4 Preparation of @ PDA @ K-UIO1/K-UIO2@ AuNPs nanoparticles:
at 250mL threeAdding 98mL of secondary water and 2mL of 50mmol/L chloroauric acid solution into the neck flask, fully stirring, putting the three-neck flask into an oil bath pot at 110 ℃ to boil for 5min, and continuously magnetically stirring; rapidly adding 10mL of sodium citrate solution with the concentration of 38.8mmoL/L after the liquid in the bottle is boiled, continuously boiling and refluxing for 20min after the color of the reaction liquid is changed from light yellow to wine red, stopping heating, stirring and cooling to room temperature, and filtering by adopting a filter membrane with the diameter of 0.22 mu m to obtain nano gold (AuNPs) solution with the particle diameter of 20-30 nm; fe obtained in the step (2) 3 O 4 Adding the @ PDA @ K-UIO1/K-UIO2 nano particles into the nano gold solution, standing overnight, removing the excessive nano gold solution on the upper layer after magnetic separation, and obtaining Fe 3 O 4 The nano particles of @ PDA @ K-UIO1/K-UIO2@ AuNPs.
(4) Preparation of aptamer fluorescent probes
Taking an aptamer DNA chain with a sulfhydryl group at the 5' -end (the sequence is 5' -SH-C6-CCACCAACGAGAGTCAGAAAACCATGGTGGG-3 ') and a freeze-drying reagent of complementary short-chain cDNA of modified fluorescein (5 ' -FAM-GGTTTTCTGAC-3 '), respectively placing the freeze-drying reagent in a centrifugal machine at room temperature, centrifuging for 5min at a speed of 10000 rpm, and then adding ultrapure water to prepare an aptamer with a concentration of 100 mu M and a fluorescent complementary strand solution; respectively activating the two solutions in a constant-temperature water bath at 90 ℃ for 10min, and rapidly transferring the two solutions into an ice-water mixture at 0 ℃ for cooling for 10min to obtain a stock solution of the aptamer and a fluorescence complementary strand thereof; preparing PBS buffer salt solution (10 mmol/L PBS, pH=7.50, 500 mmol/L NaCl), uniformly mixing the three according to the ratio of the aptamer to the complementary strand to the PBS buffer solution=1:1:8 (v/v/v), and incubating for 2 h at 55 ℃ to obtain the aptamer fluorescent probe solution.
(5) Preparation of an aptamer functionalized magnetic nano probe:
adding 0.6mg of Fe prepared in the step (3) into 50 mu L of the aptamer fluorescent probe solution prepared in the step (4) and having the concentration of 10 mu mol/L 3 O 4 The nano particles @ PDA @ K-UIO1/K-UIO2@ AuNPs are placed in a constant temperature mixing instrument, and are subjected to magnetic separation after shaking and dispersing for 15min at 25 ℃; then 40. Mu.L of 2mol/L of mercaptohexanol solution was removed and added to Fe 3 O 4 Dispersion of @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probeVibrating for 1h at 25 ℃ in a constant temperature mixing instrument, and then performing magnetic separation to obtain the mercapto hexanol-enclosed Fe 3 O 4 The probe is a @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe.
For the prepared Fe 3 O 4 @PDA@K-UIO1/K-UIO2@AuNPs、Fe 3 O 4 @PDA@K-UIO 1/K-UIO2@AuNPs@aptamer probe and mercapto hexanol blocked Fe 3 O 4 Dynamic Light Scattering (DLS) measurement is carried out on the probe of the@PDA@K-UIO 1/K-UIO2@AuNPs@aptamer, and the result shows that the molecular particle sizes of the probe are 261nm, 266nm and 273nm respectively, and the molecular particle sizes are close to the theoretical value 271.2nm of the upright distribution of the aptamer DNA chain, so that the upright distribution state of the aptamer on the MOF surface is obviously improved.
Example 2
Fe prepared by example 1 3 O 4 Detecting field halichondrin (OA) by a @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe, specifically taking the Fe 3 O 4 0.60mg of a @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe is added with 40 mu L of a field halichondrin OA solution with different concentrations, and after incubation for 120min at room temperature, supernatant is collected by magnetic separation, and a sample fluorescence signal is detected; the fluorescence measurement conditions were: laser Induced Fluorescence (LIF) excitation wavelength is 497nm, emission wavelength is 512nm, quartz capillary specification is 100 μm×500mm, mobile phase: PBS buffer ph=7.5, flow rate: 0.2 mL/min, sample volume was measured: 0.06-0.1. Mu.L of PBS buffer consisting of 10mmol/L NaH 2 PO 4 、10 mmol/L Na 2 HPO 4 500 mmol/L NaCl. The results are shown in FIGS. 2 and 3.
As can be seen from fig. 2 and 3, under the above measurement conditions, the fluorescence response intensity of the magnetic nanoprobe sensing system increases with the increase of OA concentration, and it has a good linear range (R 2 =0.997), the detection limit was calculated to be 15 ng/L according to the 3-fold standard deviation method.
The Fe prepared in example 1 was used according to the above test conditions 3 O 4 The detection results of the ligand probe @ PDA @ K-UIO1/K-UIO2@ AuNPs @ are shown in FIG. 4, wherein the ligand probe is used for detecting 1 ng/mL of okadaic acid in the field and 20ng/mL of saxitoxin, tetrodotoxin and alginic acid.
As can be seen from fig. 4, the detection specificity of the aptamer magnetic nanoprobe based on the multilayer organic metal framework-nano Jin Jie guide is good, the fluorescence intensity of the aptamer magnetic nanoprobe based on the multilayer organic metal framework-nano Jin Jie guide is kept above 120000 (a.u.), and the fluorescence intensity of other toxins is only 1000-2000 (a.u.) under the condition that the concentration of the other toxins is 20 times of that of the OA, so that the fluorescence sensing system based on the multilayer organic metal framework-nano Jin Jie guide has good anti-interference effect.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
1. An aptamer functionalized magnetic nanoprobe based on a multilayer organic metal framework-nanometer Jin Jie guide, which is characterized in that: coating magnetic nano particles with polydopamine, forming a multilayer organic metal framework-nano gold composite material by in-situ growth on the magnetic nano particles, bonding a proper ligand fluorescent probe by taking the magnetic nano particles loaded with the composite material as a carrier, and sealing by adopting a sulfhydryl reagent to form the aptamer functionalized magnetic nano probe;
the multi-layer organic metal frame-nano gold composite material is formed by bonding a multi-layer structure organic metal frame formed by two in-situ growth and central metal ion passivation on polydopamine coated magnetic nano particles with nano gold; the aptamer fluorescent probe is formed by hybridization of a nucleic acid aptamer DNA chain with a sulfhydryl group at the 5 'end and a cDNA complementary chain with a fluorescent marking group at the 5' end.
2. An aptamer functionalized magnetic nanoprobe based on a multilayer organometallic framework-nano Jin Jie guide according to claim 1, wherein the aptamer functionalized magnetic nanoprobe comprises: the two in-situ growth is specifically to fix zirconium (IV) metal center ions by utilizing amino groups of polydopamine, perform primary in-situ growth of an organic metal framework material by taking terephthalic acid as a ligand to form amino UIO-66, and perform secondary in-situ growth of the organic metal framework material by taking the obtained amino UIO-66 as a seed crystal;
the passivation of the central metal ion is to make the product after two in-situ growth and N α ,N α -passivating the bis (carboxymethyl) -L-lysine hydrate to form a lysinated product.
3. An aptamer functionalized magnetic nanoprobe based on a multilayer organometallic framework-nano Jin Jie guide according to claim 1, wherein the aptamer functionalized magnetic nanoprobe comprises: the aptamer DNA chain with the sulfhydryl group at the 5 'end is an aptamer for resisting field halichondrin, and the structural sequence is 5' -SH-C 6 CCACCAACGAGAGTCAGAAAACCATGGTGGG-3'; the structural sequence of the cDNA complementary strand with the fluorescent labeling group at the 5' end is 5' -FAM-GGTTTTCTGAC-3'.
4. An aptamer functionalized magnetic nanoprobe based on a multilayer organometallic framework-nano Jin Jie guide according to claim 1, wherein the aptamer functionalized magnetic nanoprobe comprises: the sulfhydryl reagent is sulfhydryl hexanol.
5. A method for preparing the aptamer functionalized magnetic nanoprobe based on multilayer organic metal framework-nano Jin Jie guide as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) Preparation of polydopamine coated magnetic nanoparticles:
8.1g of ferric trichloride hexahydrate is weighed and dissolved in 200mL of ethylene glycol to prepare a solution A; adding 12.0g of anhydrous sodium acetate and 2.0g of sodium citrate dihydrate into the solution A, and stirring and dissolving until a uniform and stable solution B is formed; transferring the obtained solution B into a high-pressure reaction kettle with a polytetrafluoroethylene lining, and placing the high-pressure reaction kettle into a baking oven with the temperature of 200 ℃ for reaction for 10 hours; after the reaction kettle is naturally cooled to room temperature, separating out precipitate, alternately cleaning with secondary water and ethanol for three times, and vacuum drying at 60 ℃ for 24 hours to obtain Fe 3 O 4 Magnetic nanoparticle powder; weighing Fe 3 O 4 200mg of magnetic nanoparticle powder, 240mg of dopamine hydrochloride and 240mg of tris (hydroxymethyl) aminomethane, dispersing the powder in 200mL of ultrapure water under ice water bath in an ultrasonic manner, stirring and reacting for 24h at room temperature, and thenWashing the reaction product with secondary water for multiple times to obtain polydopamine coated magnetic nano particle Fe 3 O 4 @PDA；
(2) Lysinized Fe 3 O 4 Preparation of @ PDA @ UIO/UIO2 nanoparticles:
weighing Fe 3 O 4 PDA 100 mg, zirconium chloride 71.4 mg, 2-amino terephthalic acid 50.78 mg, acetone 70 mL and acetic acid 4.2 mL, after being fully and uniformly mixed, ultrasonic treated for 10min, transferred into a polytetrafluoroethylene lining high-pressure reaction kettle, reacted for 24h at 100 ℃, washed and dried to obtain Fe 3 O 4 Pda@uio1; the prepared Fe 3 O 4 Adding the @ PDA @ UIO1 into the reaction kettle again, continuously reacting for 24 hours under the same growth concentration and reaction condition, and alternately cleaning with water and ethanol for 3 times to obtain Fe 3 O 4 @PDA@UIO1/UIO2; weigh 2 mg Fe 3 O 4 @PDA@UIO1/UIO2, dispersed in 200. Mu.L of 0.02 mol/L N α ,N α After reaction of the bis (carboxymethyl) -L-lysine hydrate solution at room temperature for 30min, the product was washed with secondary water to give Fe 3 O 4 @PDA@K-UIO1/K-UIO2 nanoparticles;
（3）Fe 3 O 4 preparation of @ PDA @ K-UIO1/K-UIO2@ AuNPs nanoparticles:
98mL of secondary water and 2mL of 50mmol/L chloroauric acid solution are added into a 250mL three-necked flask, and after the mixture is fully stirred, the three-necked flask is placed into an oil bath pot at 110 ℃ to be boiled for 5min; after the liquid in the bottle is boiled, 10mL of 38.8mmoL/L sodium citrate solution is rapidly added under the magnetic stirring condition, after the color of the reaction liquid is changed from light yellow to wine red, the reaction liquid is continuously boiled and refluxed for 20 minutes, heating is stopped, stirring and cooling are carried out to room temperature, and a filter membrane with the size of 0.22 mu m is adopted for filtering, so as to obtain AuNPs solution; adding Fe obtained in the step (2) 3 O 4 The @ PDA @ K-UIO1/K-UIO2 nano particles are placed overnight, and after the excessive nano gold solution on the upper layer is removed by magnetic separation, fe is obtained 3 O 4 The nano particles of @ PDA @ K-UIO1/K-UIO2@ AuNPs;
(4) Preparation of aptamer fluorescent probes
Taking a nucleic acid aptamer DNA strand with a sulfhydryl group at the 5 'end and a cDNA complementary strand freeze-drying reagent with a fluorescent labeling group at the 5' end, centrifuging at a speed of 10000 rpm for 5min at room temperature, and then adding ultrapure water to prepare an aptamer solution and a fluorescent complementary strand solution with a concentration of 100 mu M respectively; activating the two solutions in a constant-temperature water bath at 90 ℃ for 10min, and rapidly transferring the two solutions into an ice-water mixture at 0 ℃ for cooling for 10min to obtain an aptamer stock solution and a fluorescence complementary strand stock solution; uniformly mixing the obtained aptamer stock solution, the fluorescent complementary strand stock solution and 10mmol/L PBS buffer salt solution, and incubating at 55 ℃ for 2 h to obtain an aptamer fluorescent probe solution;
(5) Preparation of an aptamer functionalized magnetic nano probe:
adding 0.6mg of Fe prepared in the step (3) into 50 mu L of 10 mu mol/L aptamer fluorescent probe solution 3 O 4 The nano particles @ PDA @ K-UIO1/K-UIO2@ AuNPs are subjected to vibration dispersion for 15min at 25 ℃ and then subjected to magnetic separation; then 40. Mu.L of 2mol/L of mercaptohexanol solution was removed and added to Fe 3 O 4 In dispersion liquid of @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe, vibrating for 1h at 25 ℃ and then performing magnetic separation to obtain mercapto hexanol-blocked Fe 3 O 4 The probe is a @ PDA @ K-UIO1/K-UIO2@ AuNPs @ aptamer probe.
6. The method for preparing the aptamer functionalized magnetic nanoprobe based on multilayer organic metal framework-nano Jin Jie guide, which is characterized by comprising the following steps of: and (3) when the step (4) is mixed, the volume ratio of the aptamer stock solution to the fluorescence complementary strand stock solution to the PBS buffer salt solution is 1:1:8.
7. Use of an aptamer functionalized magnetic nanoprobe based on a multilayer organometallic framework-nano Jin Jie guide according to claim 1 for detecting halichondrin.
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