CN111410950B - Double-block DNA modified upconversion nanoparticle and preparation method and application thereof - Google Patents
Double-block DNA modified upconversion nanoparticle and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a diblock DNA modified upconversion nanoparticle and a preparation method and application thereof, belonging to the technical field of nano materials, wherein the diblock DNA modified upconversion nanoparticle comprises an upconversion nanoparticle and diblock DNA adsorbed on the upconversion nanoparticle; the double-block DNA comprises a poly cytosine block and a functional block; the poly cytosine block is adsorbed on the surface of the up-conversion nanoparticle, and the functional block DNA is dissociated on the surface of the up-conversion nanoparticle. The DNA modified up-conversion nano-particles obtained by the invention have good stability and monodispersity in aqueous solution, and the modification process is simple. The double-block DNA modified upconversion nanoparticle effectively combines the characteristics of upconversion nanoparticles and DNA in respective fields, and has wide application prospects in the fields of precise assembly of nanomaterials, biological targeted therapy and the like.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a double-block DNA modified upconversion nanoparticle and a preparation method and application thereof.
Background
The rare earth ion doped up-conversion nanoparticles follow a nonlinear luminescence process, have the characteristics of low biotoxicity, strong penetration depth, photobleaching resistance, no-organism background autofluorescence and the like, and are widely applied to the fields of biological imaging, targeted drug loading, biological sensing and the like. In general, small and uniform upconversion nanoparticles are synthesized in an organic solvent, and the surface of the nanoparticle contains a layer of hydrophobic ligand (usually oleic acid), which greatly limits the application of the nanoparticle in the fields of biological medicine and the like. Therefore, the phase transfer of the upconversion nanoparticles from the organic phase to the aqueous phase is realized, which becomes an important precondition for expanding more applications of the upconversion nanoparticles.
DNA, one of the life genetic materials, has good biocompatibility, and has been widely used in the fields of bio-drug loading, medical imaging, nano-assembly, etc. due to its excellent stability, easy synthesis, programmable capability, accurate distance control, and strict adherence to the base complementary pairing principle. In recent years, some researchers have focused on the surface modification of upconversion nanoparticles by DNA modification, and currently, the surface modification of upconversion nanoparticles by DNA can be mainly divided into two methods: ligand exchange and intermediaries. The DNA surface modification of the upconversion nanoparticles based on ligand exchange mainly utilizes the positive charges of rare earth doped in the upconversion nanoparticles and the negative charges carried by the DNA to carry out positive and negative electric attraction, so that the DNA replaces the ligand (such as oleic acid) of the upconversion nanoparticles, and the DNA-modified upconversion nanoparticles are obtained. Ligand exchange has the advantages that the modification process is simple, a complex chemical reaction process is avoided, the appearance of the nanoparticles cannot be damaged, the limitation is obvious, the nanoparticle is easily seriously agglomerated due to the ligand exchange between an organic phase and an aqueous phase, and the subsequent assembly of the particles can be influenced due to the disordered DNA chain segments attached to the surfaces of the nanoparticles. The surface modification of the upconversion nanoparticles based on the DNA induced by the mediator is mainly to coat a layer of material containing functional groups, such as phospholipid molecules and silicon dioxide, on the surface of the upconversion nanoparticles, so that the material is transferred from an organic phase to a water phase, and the functional groups, such as carboxyl, sulfydryl and the like, are exposed at the tail end. And then the DNA modified up-conversion nano-particles are obtained through the reaction between the DNA with modification such as amino and functional groups. The method has the advantages that the DNA modified nanoparticles have better dispersibility, but the modification process is too complicated, and the introduction of a new medium can greatly weaken the luminescence of the nanoparticles, thereby limiting the wide application of the method. Therefore, how to simply and efficiently prepare a DNA-modified upconversion nanoparticle with good monodispersity and stability remains a considerable and pending problem.
Disclosure of Invention
In view of the above, the present invention provides a double-block DNA modified upconversion nanoparticle, and a preparation method and an application thereof. The invention realizes the conversion of the up-conversion nano particles from an organic phase to a water phase by using the DNA, and better exerts the application of the DNA and the up-conversion nano particles in the field of functional biological nano.
The invention provides a double-block DNA modified up-conversion nanoparticle, which comprises an up-conversion nanoparticle and double-block DNA adsorbed on the up-conversion nanoparticle; the double-block DNA comprises a poly cytosine block and a functional block; the poly cytosine block is adsorbed on the surface of the up-conversion nanoparticle, and the functional block DNA is dissociated on the surface of the up-conversion nanoparticle.
Preferably, the number of cytosines in the poly-cytosine block is 5 to 20.
Preferably, the poly cytosine block is located at the 3 'end or the 5' end of the diblock DNA.
The invention provides a preparation method of double-block DNA modified upconversion nanoparticles, which comprises the following steps:
1) Washing the upconversion nanoparticles with hydrochloric acid to obtain upconversion nanoparticles without ligands on the surface;
2) Mixing the up-conversion nanoparticles without the ligand on the surface with a double-block DNA solution to obtain a mixed solution;
3) Successively adding 10 XTB-Na to the mixture + Buffering solution to make the final concentration of NaCl in the system be 0.08-0.12 mol/L; and then incubation is carried out to obtain the diblock DNA modified upconversion nano-particle.
Preferably, the concentration of the hydrochloric acid in the step 1) is 0.08 to 0.12mol/L, and the number of times of washing is 1 to 3 times.
Preferably, the mole ratio of the surface ligand-free up-conversion nanoparticles to the diblock DNA in step 2) is 1.
Preferably, the mixing in step 2) is dropwise adding the surface ligand-free up-conversion nanoparticles into the diblock DNA, and the mixing is accompanied by ultrasonic treatment.
Preferably, the 10 XTB-Na in step 3) + The adding times of the buffer solution are 4-6 times, and the 10 XTB-Na is added every two adjacent times + The time interval of the buffer solution is 50-70 min.
Preferably, the incubation time in step 3) is 8-14 h.
The invention also provides application of the diblock DNA modified upconversion nanoparticle in precise assembly of the nanoparticle or preparation of targeted drugs.
The invention has the beneficial effects that: the invention provides a double-block DNA modified up-conversion nanoparticle, which comprises an up-conversion nanoparticle and double-block DNA adsorbed on the up-conversion nanoparticle; the double-block DNA comprises a poly cytosine block and a functional block; due to the characteristic adsorption of the upconversion nanoparticles to cytosine C, the poly-cytosine block can be preferentially adsorbed on the surface of the upconversion nanoparticles, so that the direction of DNA modification on the surface of the upconversion nanoparticles is effectively controlled, and the functional block in the double-block DNA is dissociated on the surface of the upconversion nanoparticles for subsequent hybridization. The DNA modified upconversion nanoparticles obtained by the method have good stability and monodispersity in aqueous solution, and the modification process is simple. The double-block DNA modified upconversion nanoparticle effectively combines the characteristics of upconversion nanoparticles and DNA in respective fields, and has wide application prospects in the fields of precise assembly of nanomaterials, biological targeted therapy and the like.
The preparation method provided by the invention has the advantages of simple and mild process, does not damage the appearance and optical properties of the upconversion nanoparticles, and does not need other chemical modification on the used DNA. The preparation method is suitable for modifying the DNA on the surface of most rare earth ion-doped up-conversion nanoparticles, is suitable for modifying the DNA on the surface of up-conversion nanoparticles with different particle sizes, morphologies and luminescent properties, and has certain universality.
Drawings
FIG. 1 is a schematic structural diagram of a diblock DNA modified upconversion nanoparticle of the present invention;
FIG. 2 shows the upconversion nanoparticles (NaYF) before and after modification of DNA in the present invention 4 18 Yb,2 Er) by 18%;
FIG. 3 shows upconversion nanoparticles (NaYF) before and after modification of DNA in the present invention 4 18 Yb,2 Er) by 18%;
FIG. 4 shows upconversion nanoparticles (NaYF) before and after modification of DNA in the present invention 4 18 Yb,2 Er) by 18%;
FIG. 5 shows upconversion nanoparticles (NaYF) before and after modification of DNA in the present invention 4 18 Yb,2 Er) by 18); wherein a is oleic acid-coated up-conversion nanoparticles (OA-UCNPs), b is ligand-free UCNPs, and c is DNA-UCNPs;
FIG. 6 shows modified DNA of the present invention as upconversion nanoparticles (NaYF) 4 18 Yb,2 Er) by agarose gel electrophoresis;
FIG. 7 shows modified DNA of the present invention as upconversion nanoparticles (NaYF) 4 18 Yb,2 Er) assembled with gold nanoparticles by DNA base complementary pairing;
FIG. 8 shows modified DNA of the present invention as upconversion nanoparticles (NaYF) 4 20% Yb,5% Tm) in agarose gel electrophoresis;
FIG. 9 shows modified DNA of the present invention as upconversion nanoparticles (NaYF) 4 20 Yb, 5. Sup. Th Tm) and gold nanoparticles assembled by base complementary pairing of DNA.
Detailed Description
The invention provides a double-block DNA modified up-conversion nanoparticle, which comprises an up-conversion nanoparticle and double-block DNA adsorbed on the up-conversion nanoparticle; the double-block DNA comprises a poly cytosine block and a functional block; the poly cytosine block is adsorbed on the surface of the up-conversion nanoparticle, and the functional block DNA is dissociated on the surface of the up-conversion nanoparticle.
In the present invention, the number of cytosines in the poly-cytosine block is preferably 5 to 20, and the number of cytosines is preferably set according to specific assembly requirements. In the present invention, the poly cytosine block may be located at the 3 'end or the 5' end of the diblock DNA. The invention has no special requirements on the specific sequence and length of the functional block and can be designed according to actual requirements.
In the present invention, the upconversion nanoparticles are preferably NaMF 4 :Yb 3+ /Ln 3+ Rare earth ion doped upconversion nanoparticles, wherein M = Y, gd or Sc; ln = Er or Tm; in the specific implementation process of the invention, the upconversion nanoparticles can be NaYF 4 :Yb/Er，NaYF 4 Yb/Tm or NaScF 4 Yb/Er; in the present invention, the preferred surface of the upconversion nanoparticle contains oleic acid ligands.
The invention also provides a preparation method of the double-block DNA modified upconversion nanoparticle, which comprises the following steps: 1) Washing the up-conversion nanoparticles with hydrochloric acid to obtain up-conversion nanoparticles without ligands on the surface; 2) Mixing the up-conversion nanoparticles without the ligand on the surface with a double-block DNA solution to obtain a mixed solution; 3) Successively adding 10 XTB-Na to the mixture + Buffering solution to make the final concentration of NaCl in the system be 0.08-0.12 mol/L; then incubation is carried out to obtain diblock DNA-modified upconversion nanoparticles.
In the present invention, washing the upconversion nanoparticles with hydrochloric acid yields upconversion nanoparticles with no ligand on the surface. In the present invention, the concentration of the hydrochloric acid is preferably 0.08 to 0.12mol/L, more preferably 0.1mol/L; the number of washing is preferably 1 to 3, more preferably 2. In the present invention, the cleaning process is preferably accompanied by ultrasonic treatment, and the power of the ultrasonic treatment is preferably 80 to 120w, and more preferably 100w. In the specific implementation process of the invention, the cleaning comprises the following steps: s1) dispersing 10mg of up-conversion nanoparticles into absolute ethyl alcohol, centrifuging, and collecting a first precipitate; s2) dispersing the collected first precipitate in absolute ethyl alcohol, mixing with hydrochloric acid, cleaning, and centrifuging to obtain a second precipitate; s3) dispersing the second precipitate in absolute ethyl alcohol, mixing with hydrochloric acid, cleaning, and centrifuging to obtain a third precipitate; and S4) dispersing the third precipitate in ultrapure water to obtain the upconversion nanoparticles with the surfaces free from ligands.
In the present invention, the ratio of the upconversion nanoparticles to absolute ethanol in step S1) is preferably 10mg:1mL, wherein the rotating speed of the centrifugation in the step S1) is preferably 14000-16000 rpm, and more preferably 15000rpm; the time for centrifugation is preferably 1-3 min, more preferably 2min; the volume ratio of the absolute ethyl alcohol to the hydrochloric acid in the step S2) is preferably 1.5-2.5, more preferably 2:1, and the time of the ultrasonic treatment in the step S2) is preferably 30S; the rotation speed of the centrifugation in the step S2) is preferably 14000-16000 rpm, more preferably 15000rpm; the time for the centrifugation is preferably 12 to 18min, more preferably 15min. The volume ratio of the absolute ethyl alcohol to the hydrochloric acid in the step S3) is preferably 4-6:1, and more preferably 5:1; the time of the ultrasound in the step S3) is preferably 10S, and the rotating speed of the centrifugation in the step S3) is preferably 14000-16000 rpm, more preferably 15000rpm; the time for the centrifugation is preferably 12 to 18min, more preferably 15min. In the present invention, the concentration of the surface ligand-free upconversion nanoparticles obtained in step S4) is preferably 0.06 to 0.065nmol/mL, more preferably 0.0633nmol/mL.
After the surface ligand-free up-conversion nano-particles are obtained, the surface ligand-free up-conversion nano-particles are mixed with a double-block DNA solution to obtain a mixed solution. In the present invention, the mole ratio of the surface ligand-free up-conversion nanoparticle to the diblock DNA is preferably 1. In the present invention, the concentration of the diblock DNA solution is preferably 65 to 70nmol/mL, more preferably 66.67nmol/mL. In the invention, the mixing is to add the upconversion nanoparticles without ligand on the surface into the diblock DNA dropwise, and the mixing is accompanied by ultrasonic treatment, wherein the power of the ultrasonic treatment is preferably 80-120 w, and more preferably 100w. In the present invention, after the completion of the mixing, the ultrasonic treatment is preferably continued for 20 to 40min, more preferably for 30min.
After the mixed solution is obtained, the 10 XTB-Na is added into the mixed solution successively + Buffering solution to make the final concentration of NaCl in the system 0.08-0.12 mol/L; and then incubation is carried out to obtain the diblock DNA modified upconversion nano-particle. In the invention, the 10 XTB-Na + The number of times of addition of the buffer solution is preferably 4 to 6 times, more preferably 5 times, and the 10 XTB-Na is added twice adjacent to each other + The time interval of the buffer solution is preferably 50 to 70min, more preferably 60min. In the present invention, the final concentration of NaCl in the system is preferably 0.1mol/L; in the practice of the present invention, 10 XTB-Na is preferably added to the mixture in 5 portions + Buffering solution to make NaCl concentration in the system be 0.02mol/L, 0.04mol/L, 0.06mol/L, 0.08mol/L and 0.1mol/L in sequence, and making the final system be in 1 XTB-Na + Incubation was performed in ambient. In the invention, the 10 XTB-Na + The buffer solution is 10 XTB-Na in 1L by taking water as a solvent + The buffer solution preferably comprises the following components: 890mM tris (hydroxymethyl) aminomethane (tris base), 890mM boric acid (bonic acid), 1M sodium chloride (NaCl); the 10 XTB-Na + The pH of the buffer solution is preferably 8.
In the present invention, the incubation time is preferably 8 to 14 hours, and more preferably 12 hours. After the incubation, the invention preferably further comprises the steps of filtering and centrifuging by a filter membrane; the pore size of the filter membrane is preferably 0.22 μm; the filter membrane filtration is used for removing agglomerated particles in the solution; after the filtration of the filter membrane, collecting filtrate for centrifugation, wherein the rotation speed of the centrifugation is preferably 8000-12000 rpm, more preferably 10000rpm, and the time of the centrifugation is preferably 12-18 min, more preferably 15min; the centrifugation is used for removing redundant DNA in the filtrate; and after the centrifugation is finished, removing the supernatant, adding ultrapure water or buffer solution, and repeatedly centrifuging for 3-5 times until the supernatant does not contain DNA. In the present invention, it is preferable to detect the supernatant after centrifugation by ultraviolet absorption spectroscopy until the characteristic absorption of DNA at 260nm cannot be detected in the supernatant.
The invention also provides application of the diblock DNA modified upconversion nanoparticle in precise assembly of the nanoparticle or preparation of targeted drugs. The preparation method of the double-block DNA modified up-conversion nanoparticles provided by the invention is suitable for surface DNA modification of up-conversion nanoparticles with different particle sizes, shapes and optical properties, so that the direction of DNA on the surface of the up-conversion nanoparticles is controllable, the obtained DNA modified up-conversion nanoparticles have good stability and monodispersity in aqueous solution, and can be used for precise assembly between the nanoparticles and DNA origami and applied to the preparation process of targeted drugs.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Hexagonal surface phase NaYF 4 18% Yb,2% Er Up-converting nanoparticles DNA modification and Assembly of DNA-modified Up-converting nanoparticles with gold nanoparticles (5nm, DNA-AuNPs)
Hexagonal phase NaYF 4 18% Yb,2% preparation of Er upconversion nanoparticles: weighing 0.8mmol of anhydrous YCl 3 0.18mmol of anhydrous YbCl 3 And 0.02mmol of Anhydrous ErCl 3 7.5mL of Oleic Acid (OA) and 17.5mL of Octadecene (ODE) were weighed out and added together to a 50mL three-necked round-bottomed flask. The reflux condenser was opened and nitrogen was rapidly introduced, and the round-bottomed flask was placed in a heating mantle, heated to 150 ℃ and stirred at a constant temperature for 30min, followed by cooling to room temperature. The reflux condenser was removed and the contents 2 were added to the round bottom flask.5mmol NaOH and 5.5mmol NH 4 And F, heating the methanol solution to 100 ℃, and stirring at constant temperature for 30min. After that, the reaction mass was warmed up to 300 ℃ and reacted at constant temperature for 1h, and finally cooled to room temperature. Centrifuging the obtained reactant at 6000rpm for 5min, taking the lower layer precipitate, washing the obtained product twice with cyclohexane and ethanol to obtain hexagonal phase NaYF 4 18% Yb,2% Er upconversion nanoparticles, stored in cyclohexane for future use.
Preparation of diblock DNA-modified upconversion nanoparticles:
removing the oleic acid ligand on the surface of the upconversion nanoparticles:
(1) 10mg of upconversion nanoparticles with oleic acid ligand are dispersed in 1mL of absolute ethyl alcohol and centrifuged under the conditions of the rotating speed of 15000rpm and the time of 2min to obtain lower-layer precipitate.
(2) Adding 1mL of absolute ethanol and 500. Mu.L of 0.1mol/L hydrochloric acid to the lower layer precipitate obtained in (1), subjecting to ultrasonic treatment (power 100W) for 30s, centrifuging at 15000rpm for 15min, and collecting the lower layer precipitate.
(3) Adding 1mL of absolute ethyl alcohol and 20 mu L of 0.1mol/L hydrochloric acid into the lower-layer sediment obtained in the step (2), then carrying out ultrasonic treatment (with the power of 100W) for 10s, centrifuging at the rotating speed of 15000rpm for 15min, taking the lower-layer sediment, obtaining the ligand-free up-conversion nanoparticles (ligand-free UCNPs) on the surface, and dispersing in 1mL of ultrapure water for later use.
DNA-modified upconversion nanoparticles
0.038nmol of ligand-free surface up-conversion nanoparticle ligand-free UCNPs were dispersed in 600. Mu.L of ultrapure water, and an aqueous solution of ligand-free UCNPs was slowly dropped into 300. Mu.L of ultrapure water containing 20nmol of diblock DNA (5'-CTAAGACTATGTGGACCCCCCCCCCCCCCC-3', SEQ ID No. 1) under the ultrasonic condition, and the obtained mixed solution was continued to be subjected to ultrasonic treatment for 30min. After the ultrasound was finished, 20. Mu.L of 10 XTB-Na was added + Adding buffer solution into the above mixed solution, shaking, and adding 20 μ L10 XTB-Na dropwise at an interval of 60min + The buffer solution is used for ensuring that the concentration of NaCl in the system is 0.02mol/L, 0.04mol/L, 0.06mol/L, 0.08mol/L and 0.1mol/L in sequence. At the final systemIn 1 XTB-Na + After incubation overnight (> 8 h) in the environment, the solution was passed through a 0.22 μm filter to remove agglomerated particles from the solution. And finally, centrifuging the obtained solution, and removing redundant DNA in the solution to obtain the DNA modified up-conversion nanoparticles.
Assembling DNA-UCNPs and DNA-AuNPs:
gold nanoparticles (purchased from Ted Pella, usa) were surface modified with DNA (5 '-TCCACATAGTCTTAGTTTTT-SH-3', SEQ ID No. 2) using conventional thiol DNA, see references (m.r.jones, r.j.macfarlane, b.lee, j.zhang, k.young, a.j.senesi, c.a.mirkin, nat.mater.2010,9, 913-917.). And (2) mixing the DNA modified up-conversion nanoparticles with the gold nanoparticles according to the DNA-UCNPs: DNA-AuNPs =1, 50 molar ratio mixed and dispersed in 1 xtb-Na + And (3) in a buffer solution, placing the system in a constant-temperature metal bath, shaking up for 5h at 45 ℃ with mild shaking, naturally cooling to room temperature, and centrifuging to remove unassembled gold nanoparticles to obtain the UCNPs @ DNA assembly.
10×TB-Na + Buffer solution: 890mM tris (base), 890mM boric acid (bonic acid), 1M sodium chloride (NaCl), pH =8.
1×TB-Na + Buffer solution: 89mM tris (hydroxymethyl) aminomethane (tris base), 89mM boric acid (bonic acid), 0.1M sodium chloride (NaCl), pH =8.
FIG. 2 shows up-conversion nanoparticles (NaYF) before and after DNA modification 4 18 Yb,2 Er) by 18%. According to UV, ligand-free UCNPs have no characteristic absorption peak at 260nm, and UCNPs after DNA modification have obvious characteristic absorption peak of DNA at 260nm, which indicates that DNA is successfully modified on the surface of the UCNPs. The ultraviolet absorption spectrum measured in the experiment is characterized in that a sample is placed in a 2mL quartz cuvette and is measured by a UV-1800 ultraviolet visible spectrophotometer.
FIG. 3 shows up-conversion nanoparticles (NaYF) before and after DNA modification 4 18 Yb,2 Er) in a DLS spectrum. DLS shows that the particle size of UCNPs is increased from 25nm to 35nm due to the modification of surface DNA. The DLS spectrogram obtained in the experiment is characterized in that a sample is placed in a 2mL quartz cuvette and is characterized under a nanometer particle size and potential analyzer。
FIG. 4 shows up-conversion nanoparticles (NaYF) before and after DNA modification 4 18 Yb,2 Er) by 18% by weight. Due to the surface protonation, UCNPs which are removed from the surface ligand by using hydrochloric acid are electropositive, a DNA skeleton is electronegative, and the surface of the DNA modified upconversion nanoparticle is electronegative. The Zeta spectrogram obtained in the experiment is characterized in that a sample is placed in a Marvin disposable folding capillary Zeta potential sample pool and is characterized under a nanometer particle size and potential analyzer.
FIG. 5 shows up-converting nanoparticles (NaYF) before and after DNA modification 4 18 Yb,2 Er). According to TEM, the morphology and the size of the upconversion nanoparticles before and after DNA modification are not obviously changed, which shows that the method provided by the invention is a mild method for modifying the upconversion nanoparticles by DNA, and the morphology and the size of the nanoparticles cannot be changed. The preparation method of the TEM sample in the experiment comprises the following steps: a small amount of sample was dropped on a common carbon supported film copper mesh and the sample was allowed to dry at room temperature. And (3) carrying out appearance, size and other representations on the sample on the copper mesh by using a 120kV biological transmission electron microscope.
FIG. 6 is a DNA-modified upconversion nanoparticle (NaYF) 4 18 Yb,2 Er) in the sample. As shown in FIG. 6, the DNA-UCNPs showed a regular band in 1% agarose gel electrophoresis, which indicates that the DNA-UCNPs obtained by the present invention have good stability in aqueous solution. Preparation of agarose gel in the experiment: 0.6g of agarose was weighed into a beaker, and 60mL of 1 XTB buffer solution (89 mM Tris (tris base), 89mM boric acid (boric acid) pH = 8) and 1. Mu.L of gel red (staining of DNA) were added thereto, and the beaker was placed in a microwave oven and heated to prepare a 1% agarose gel. And adding the sample into the gel hole, so that the sample is transferred from the negative electrode to the positive electrode, and finally characterizing the sample by using a gel imager.
FIG. 7 shows DNA-modified upconversion nanoparticles (NaYF) 4 18% Yb,2% Er) by DNA with gold nanoparticles TEM image after base complementary pairing assembly. Therefore, the DNA on the surface of the UCNPs can still keep the original base complementary pairing property, and can be applied to the assembly of nano-particles. TEM sample preparation procedureThe same procedure as described for the preparation of fig. 5.
Example 2
Synthesis of hexagonal phase NaYF 4 20% Yb,5% Tm upconversion nanoparticles: weighing 0.75mmol of anhydrous YCl 3 0.2mmol of anhydrous YbCl 3 And 0.05mmol of anhydrous TmCl 3 7.5mL of Oleic Acid (OA) and 17.5mL of Octadecene (ODE) were weighed out and added together to a 50mL three-necked round-bottomed flask. The reflux condenser was opened and nitrogen was rapidly introduced, and the round-bottomed flask was placed in a heating mantle, heated to 150 ℃ and stirred at a constant temperature for 30min, followed by cooling to room temperature. After taking down the reflux condenser, a round-bottomed flask was charged with a solution containing 2.5mmol of NaOH and 5.5mmol of NH 4 And F, heating to 100 ℃, and stirring at constant temperature for 30min. After that, the reaction mass was warmed up to 300 ℃ for isothermal reaction for 1.5h, and finally cooled to room temperature. Centrifuging the obtained reactant at 6000rpm for 5min, collecting the lower layer precipitate, washing the obtained product with cyclohexane and ethanol twice to obtain hexagonal phase NaYF 4 20% Yb,5% Tm upconversion nanoparticles, stored in cyclohexane for future use.
5nm gold nanoparticles were purchased from Ted Pella, USA.
Removing the oleic acid ligand on the surface of the upconversion nanoparticles:
(1) 10mg of upconversion nanoparticles with oleic acid ligand are dispersed in 1mL of absolute ethyl alcohol and centrifuged under the conditions of the rotating speed of 15000rpm and the time of 2min to obtain lower-layer precipitate.
(2) Adding 1mL of absolute ethanol and 500. Mu.L of 0.1mol/L hydrochloric acid to the lower layer precipitate obtained in (1), subjecting to ultrasonic treatment (power 100W) for 30s, centrifuging at 15000rpm for 15min, and collecting the lower layer precipitate.
(3) Adding 1mL of absolute ethyl alcohol and 20 muL of 0.1mol/L hydrochloric acid into the lower-layer sediment obtained in the step (2), then carrying out ultrasonic treatment (power of 100W) for 10s, centrifuging at the rotating speed of 15000rpm for 15min, and taking the lower-layer sediment to obtain the upconversion nanoparticles (ligand-free UCNPs) without ligands on the surface, and dispersing the upconversion nanoparticles (ligand-free UCNPs) in 1mL of ultrapure water for later use.
DNA-modified upconversion nanoparticles
0.038nmol of ligand-free surface up-conversion nanoparticle ligand-free UCNPs were dispersed in 600. Mu.L of ultrapure water, and an aqueous solution of ligand-free UCNPs was slowly dropped into 300. Mu.L of ultrapure water containing 20nmol of diblock DNA (5'-CTAAGACTATGTGGACCCCCCCCCCCCCCC-3', SEQ ID No. 1) under the ultrasonic condition, and the obtained mixed solution was continued to be subjected to ultrasonic treatment for 30min. After the ultrasound was finished, 20. Mu.L of 10 XTB-Na was added + Adding buffer solution into the above mixed solution, shaking, and adding 20 μ L10 XTB-Na dropwise at an interval of 60min + The buffer solution is used for ensuring that the concentration of NaCl in the system is 0.02mol/L, 0.04mol/L, 0.06mol/L, 0.08mol/L and 0.1mol/L in sequence. The final system is at 1 XTB-Na + After incubation overnight (> 8 h) in the environment, the solution was passed through a 0.22 μm filter to remove agglomerated particles from the solution. And finally, centrifuging the obtained solution, and removing redundant DNA in the solution to obtain the DNA modified upconversion nanoparticles.
Assembling DNA-UCNPs and DNA-AuNPs:
gold nanoparticles (purchased from Ted Pella, usa) were surface modified with DNA (5 '-TCCACATAGTCTTAGTTTTT-SH-3', SEQ ID No. 2) using conventional thiol DNA, see references (m.r.jones, r.j.macfarlane, b.lee, j.zhang, k.young, a.j.senesi, c.a.mirkin, nat.mater.2010,9, 913-917.). And (2) mixing the DNA modified up-conversion nanoparticles with the gold nanoparticles according to the DNA-UCNPs: DNA-AuNPs =1, 50 molar ratio mixed and dispersed in 1 xtb-Na + And (3) in a buffer solution, placing the system in a constant-temperature metal bath, shaking up for 5h at 45 ℃ with mild shaking, naturally cooling to room temperature, and centrifuging to remove unassembled gold nanoparticles to obtain the UCNPs @ DNA assembly.
FIG. 8 is a DNA-modified upconversion nanoparticle (NaYF) 4 20 Yb, 5. Sup. Th Tm) by agarose gel electrophoresis. As shown in FIG. 8, the DNA-UCNPs showed a regular band in 1% agarose gel electrophoresis, which indicates that the DNA-UCNPs obtained by the present invention have good stability in aqueous solution. Preparation of agarose gel in the experiment:0.6g of agarose was weighed into a beaker, 60mL of 1 XTB buffer solution (89 mM tris base, 89mM boric acid pH = 8), 1. Mu.L of gel red (staining of DNA) was added, and the beaker was placed in a microwave oven and heated to prepare a 1% agarose gel. And adding the sample into the gel hole, so that the sample is transferred from the negative electrode to the positive electrode, and finally characterizing the sample by using a gel imager.
FIG. 9 shows DNA-modified upconversion nanoparticles (NaYF) 4 18 Yb, 2. Sup. Th Tm) and gold nanoparticles by DNA base complementary pairing. Therefore, the DNA on the surface of the UCNPs can still keep the original base complementary pairing property, and can be applied to the assembly of nano-particles. The preparation method of the TEM sample in the experiment comprises the following steps: a small amount of sample was dropped on a common carbon supported film copper mesh and the sample was allowed to dry at room temperature. And (3) carrying out appearance, size and other representations on the sample on the copper mesh by using a 120kV biological transmission electron microscope.
From the above embodiments, the preparation method of the double-block DNA modified upconversion nanoparticles provided by the present invention is suitable for surface DNA modification of upconversion nanoparticles with different particle sizes, morphologies, and optical properties, so that the direction of DNA on the surface of the upconversion nanoparticles is controllable, and the obtained DNA modified upconversion nanoparticles have good stability and monodispersity in an aqueous solution, can perform precise assembly between the nanoparticles and DNA origami, and can be applied to the preparation process of a targeted drug.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Changzhou noda Biochemical technology Co., ltd
<120> double-block DNA modified upconversion nanoparticle and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctaagactat gtggaccccc cccccccccc 30
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Claims (9)
1. A double-block DNA modified up-conversion nanoparticle is characterized by comprising an up-conversion nanoparticle and double-block DNA adsorbed to the up-conversion nanoparticle; the double-block DNA comprises a poly cytosine block and a functional block; the poly cytosine block is adsorbed on the surface of the up-conversion nanoparticle, and the functional block DNA is dissociated on the surface of the up-conversion nanoparticle;
the number of cytosines in the poly-cytosine block is 5-20.
2. The double-block DNA-modified upconversion nanoparticle according to claim 1, wherein the poly-cytosine block is located at the 3 'end or the 5' end of the double-block DNA.
3. The method for preparing the double-block DNA modified upconversion nanoparticle of claim 1 or 2, comprising the steps of:
1) Washing the up-conversion nanoparticles with hydrochloric acid to obtain up-conversion nanoparticles without ligands on the surface;
2) Mixing the up-conversion nanoparticles without the ligand on the surface with a double-block DNA solution to obtain a mixed solution;
3) Successively adding 10 XTB-Na to the mixture + Buffering solution to make the final concentration of NaCl in the system 0.08-0.12 mol/L; then incubation is carried out to obtain double-block DNA modified up-conversion nanoparticles;
the 10 XTB-Na + The buffer solution is 10 XTB-Na in 1L by taking water as a solvent + The buffer solution comprises the following components: 890mM Tris, 890mM boric acid, 1M sodium chloride;
the 10 XTB-Na + The pH of the buffer solution was 8.
4. The method according to claim 3, wherein the concentration of the hydrochloric acid in the step 1) is 0.08 to 0.12mol/L, and the number of times of the washing is 1 to 3 times.
5. The preparation method of claim 3, wherein the molar ratio of the surface ligand-free upconversion nanoparticles to the diblock DNA in step 2) is 1.
6. The preparation method according to claim 3 or 5, wherein the mixing in step 2) is dropwise adding the surface ligand-free up-conversion nanoparticles into the diblock DNA, and the mixing is accompanied by ultrasonic treatment.
7. The method according to claim 3, wherein the 10 XTB-Na is used in the step 3) + The adding times of the buffer solution are 4-6 times, and the 10 XTB-Na is added every two adjacent times + The time interval of the buffer solution is 50-70 min.
8. The method according to claim 3, wherein the incubation time in step 3) is 8 to 14 hours.
9. Use of the diblock DNA-modifying upconversion nanoparticle according to claim 1 or 2 or the diblock DNA-modifying upconversion nanoparticle prepared according to the preparation method of any one of claims 3 to 8 for precise assembly of nanoparticles or targeted drug preparation.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818325A (en) * | 2015-04-16 | 2015-08-05 | 上海大学 | Nanoprobe for acute promyelocytic leukemia fluorescence detection and preparing method thereof |
| CN105002285A (en) * | 2015-07-30 | 2015-10-28 | 中国人民解放军第三军医大学第一附属医院 | Liquid-stage chip constant-temperature detection method for tiny RNA |
| CN106596971A (en) * | 2016-12-16 | 2017-04-26 | 江南大学 | Precious metal plasma effect enhanced up-conversion fluorescence structure-based method for hypersensitive detection of vascular endothelial growth factor |
| CN108196044A (en) * | 2017-12-27 | 2018-06-22 | 西安交通大学 | A kind of effluent test paper detecting method based on up-conversion fluorescence aptamers |
| CN109187473A (en) * | 2018-09-15 | 2019-01-11 | 福建医科大学 | Fluorescence resonance energy transfer based on up-conversion nano material and tetramethylrhodamine is used for the detection of excretion body |
| CN109307664A (en) * | 2018-09-30 | 2019-02-05 | 江南大学 | The fluorescent detection probe of metal ion in a kind of detectable living cells |
| CN110296961A (en) * | 2018-03-22 | 2019-10-01 | 中国科学院上海高等研究院 | The building and application of controllable Nano-Au probe based on double block D NA |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190119731A1 (en) * | 2017-10-24 | 2019-04-25 | The Hong Kong Polytechnic University | Nanoprobe sandwich assay for nucleotide sequence detection |
-
2020
- 2020-02-25 CN CN202010118679.XA patent/CN111410950B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104818325A (en) * | 2015-04-16 | 2015-08-05 | 上海大学 | Nanoprobe for acute promyelocytic leukemia fluorescence detection and preparing method thereof |
| CN105002285A (en) * | 2015-07-30 | 2015-10-28 | 中国人民解放军第三军医大学第一附属医院 | Liquid-stage chip constant-temperature detection method for tiny RNA |
| CN106596971A (en) * | 2016-12-16 | 2017-04-26 | 江南大学 | Precious metal plasma effect enhanced up-conversion fluorescence structure-based method for hypersensitive detection of vascular endothelial growth factor |
| CN108196044A (en) * | 2017-12-27 | 2018-06-22 | 西安交通大学 | A kind of effluent test paper detecting method based on up-conversion fluorescence aptamers |
| CN110296961A (en) * | 2018-03-22 | 2019-10-01 | 中国科学院上海高等研究院 | The building and application of controllable Nano-Au probe based on double block D NA |
| CN109187473A (en) * | 2018-09-15 | 2019-01-11 | 福建医科大学 | Fluorescence resonance energy transfer based on up-conversion nano material and tetramethylrhodamine is used for the detection of excretion body |
| CN109307664A (en) * | 2018-09-30 | 2019-02-05 | 江南大学 | The fluorescent detection probe of metal ion in a kind of detectable living cells |
Non-Patent Citations (2)
| Title |
|---|
| Designed Diblock Oligonucleotide for the Synthesis of Spatially Isolated and Highly Hybridizable Functionalization of DNA−Gold Nanoparticle Nanoconjugates;Hao Pei 等;《J. Am. Chem. Soc.》;20120716;第134卷;11876-11879 * |
| Poly-cytosine DNA as a High-Affinity Ligand for Inorganic Nanomaterials;Chang Lu 等;《Angew. Chem. Int. Ed.》;20170421;第56卷;6208-6212 * |
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