CN112920421B - Temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe and preparation method and application thereof - Google Patents
Temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe and a preparation method and application thereof, wherein the preparation of the quantum dot fluorescent probe comprises the following steps: growing a silicon dioxide shell layer on the surface of the gold nano-particle to form Au @ SiO2(ii) a Isopropyl acrylamide monomer, acrylic acid, ammonium persulfate and Au @ SiO2Mixing in SiO2Surface polymerization to form SiO2Coating the surface with an isopropyl acrylamide monomer to obtain Au @ SiO2@ PNIPAM microspheres; mixing Au @ SiO2The @ PNIPAM microsphere is placed in EDC aqueous solution, and then the quantum dot modified by amino is added to carry out amidation reaction, so that the quantum dot is successfully connected with Au @ SiO2Forming Au @ SiO on the surface of the @ PNIPAM microsphere2@ PNIPAM-QDs fluorescent probes. The fluorescent probe prepared by the invention has sensitive temperature responsiveness and can be applied to temperature measurement.
Description
Technical Field
The invention belongs to the field of high molecular materials, and particularly relates to a temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe, and a preparation method and application thereof.
Background
Quantum Dots (QDs) are a new class of fluorescent nanomaterials. Because the luminous efficiency is higher, photobleaching is not easy to occur, and the monochromaticity is good, the application in biological imaging, biological sensing and fluorescence detection is more and more concerned.
The existing temperature fluorescent probe technology is more perfect, but the problems of higher preparation difficulty, narrow measurement temperature range, insensitive detection and the like still exist. Meanwhile, because the surface ligand of the water-soluble quantum dot is unstable, when the water-soluble quantum dot and the monomer are subjected to radical polymerization together, the free radical molecules can easily quench the fluorescence of the quantum dot, and therefore the water-soluble quantum dot is rarely applied to the preparation of the temperature fluorescent probe.
Therefore, the technical problems of high preparation difficulty and insensitive detection of the conventional quantum dot fluorescent probe exist.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides a temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe and a preparation method and application thereof, so that the technical problems of high preparation difficulty and insensitive detection of the conventional quantum dot fluorescent probe are solved.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing a temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe, comprising the steps of:
(1) growing a silicon dioxide shell layer on the surface of the gold nano-particle to form Au @ SiO2；
(2) Isopropyl acrylamide (NIPAM) monomer, Acrylic Acid (AA), Ammonium Persulfate (APS) and Au @ SiO2Mixing in SiO2Surface polymerization to form SiO2Coating the surface with an isopropyl acrylamide monomer to obtain Au @ SiO2@ PNIPAM microspheres;
(3) mixing Au @ SiO2The @ PNIPAM microsphere is placed in EDC (1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride) aqueous solution, then the amino modified quantum dot is added to carry out amidation reaction,so that the quantum dots are successfully connected at Au @ SiO2The reaction is carried out for 6 to 10 hours at normal temperature on the surface of the @ PNIPAM microsphere to form Au @ SiO2@ PNIPAM-QDs fluorescent probes.
Further, the step (1) includes:
mixing gold nanoparticles, tetraethoxysilane, sodium hydroxide, ethanol and water according to the volume ratio of 2000: 15: 167: 2985: 16670-2。
Further, the step (1) further comprises:
and silicon dioxide shells with different thicknesses grow on the surfaces of the gold nanoparticles by adding tetraethoxysilane with different contents.
Further, the preparation of the gold nanoparticles comprises:
in chloroauric acid (HAuCl)4) Adding water, sodium citrate and sodium borohydride into chloroauric acid to form seed liquid, and adding water, Cetyl Trimethyl Ammonium Bromide (CTAB) and ascorbic acid into chloroauric acid to form growth liquid, wherein the volume ratio of chloroauric acid to water to sodium citrate to sodium borohydride is as follows: 5: 190: 5: 6, the concentration of chloroauric acid is 10mmol/l, the concentration of sodium citrate is 2.94mg/ml, the concentration of sodium borohydride is 3.783mg/ml, and the volume ratio of chloroauric acid, water, cetyl trimethyl ammonium bromide and ascorbic acid is as follows: 6: 114: 96: 1.6, the concentration of chloroauric acid is 10mmol/1, the concentration of hexadecyl trimethyl ammonium bromide is 0.2mol/1, the concentration of ascorbic acid is 17.6mg/ml, and the seed solution is added into the growth solution to prepare the gold nano-particles.
Further, the step (1) further comprises:
at Au @ SiO2Adding a silane coupling agent, and fully reacting to obtain Au @ SiO with double bonds on the surface2. Silane coupling agent and Au @ SiO2The ratio of (A) to (B) was 2. mu.l: 3 mg. At this time, Au @ SiO2In ethanol, Au @ SiO2The ratio of ethanol to ethanol was 3 mg: 3 ml. The silane coupling agent is: 3- (methacryloyloxy) propyltrimethoxysilane.
Further, the stepsThe reaction temperature after mixing in the step (2) is 70-80 ℃, and the reaction time is 5-8 h. In the step (2), isopropyl acrylamide monomer, acrylic acid, ammonium persulfate and Au @ SiO2The mass ratio of (A) to (B) is as follows: 45: 4.5: 15: 3, and ultrapure water is also added during the reaction, and the mass of the ultrapure water is Au @ SiO21000 times the mass of (c).
In the step (3), Au @ SiO2The volume ratio of the @ PNIPAM microsphere, the EDC and the quantum dots is 3000: 200: 1-3000: 200: 100, and preferably 3000: 200: 8.
Further, the particle size of the gold nanoparticles is 1nm-100 nm.
Further, the thickness of the silicon dioxide shell layer is 2nm-50 nm.
The water used in the invention is ultrapure water, the concentration of ethanol used in the invention is more than or equal to 99.7 percent, and the concentration of sodium hydroxide is 0.1 mol/L.
According to another aspect of the invention, the invention provides a temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe, which is prepared by the preparation method of the quantum dot fluorescent probe.
According to another aspect of the invention, the invention provides an application of the temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe, wherein the quantum dot fluorescent probe is prepared by the preparation method of the quantum dot fluorescent probe, and the quantum dot fluorescent probe is applied to temperature measurement.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
(1) the invention changes the fluorescence intensity of quantum dots by utilizing the surface plasma effect of gold nanoparticles, and firstly, Au @ SiO2The surface carries out free radical reaction on N-isopropyl acrylamide and acrylic acid, and after polymerization is finished, quantum dots are connected to the surface by adopting amidation reaction, and the amidation reaction is mild, so that the fluorescence of the quantum dots cannot be obviously influenced. N-isopropylacrylamide (NIPAM) as a temperature-sensitive monomer can respond to phase change of temperature to form polymerizationThe polymer is composed of three parts of a macromolecular main chain, a hydrophobic group and a hydrophilic group. With the increase of the temperature, the molecular chain of the PNIPAM polymerized on the surface of the silicon dioxide gradually shrinks, so that the distance between the quantum dot and the gold nanoparticle is reduced, the quenching effect of the gold nanoparticle on the fluorescence of the quantum dot is increased, and the fluorescence of the quantum dot is obviously weakened, thereby achieving the purpose of detecting the temperature change with sensitivity. The fluorescent probe prepared by the invention has sensitive temperature responsiveness and can be applied to temperature measurement.
(2) Gold nanoparticles (AuNPs) as a noble metal have strong influence on the fluorescence emission intensity of QDs, and the influence process can be divided into two cases: 1. non-radiative energy transfer (NRET) from QDs to AuNPs results in fluorescence quenching. 2. Local Field Enhancement (LFE) causes fluorescence enhancement. When the distance is close, NRET plays a dominant role, causing fluorescence quenching; with increasing distance, the NRET phenomenon gradually weakens, LFE plays a dominant role, causing fluorescence enhancement; as the distance increases again, both the NRET and LFE effects diminish. Therefore, the intensity of fluorescence is greatly related to the regulation distance. Compared with a hydrothermal method, the gold nanoparticles prepared by adopting a seed growth method have mild reaction conditions, and a CTAB ligand is adopted in the preparation process, so that the obtained nanoparticles are relatively stable, and the particle size distribution uniformity is relatively good. The stober method is adopted to coat the silicon dioxide shell layer on the surface of the gold nano particle, and the prepared nano particle has better morphology uniformity because the stober method is complete in silicon growing system. Different silicon dioxide shell thicknesses can be obtained by adding different amounts of TEOS, so that the distances between the gold nanoparticles and the quantum dots are different, and the action of the gold nanoparticles on surface electron bodies of the quantum dots is closely related to the distances, thereby obtaining the quantum dots with different fluorescence quenching degrees.
(3) The invention uses NIPAM monomer, AA monomer and APS initiator to polymerize on SiO by free radical2Surface polymerization to form SiO2The surface of the material is coated with a temperature-sensitive polymer PNIPAM, and an AA monomer provides a carboxyl group, so that the material is convenient to perform amidation reaction with the amino-modified quantum dot, and the quantum dot can be successfully connected with Au @ SiO2@ PNIPAM microsphere surface. At the same time, the free radical reaction is conductedThe operation is simple. According to the invention, double bonds are introduced on the surface of a silica shell layer, so that NIPAM can be polymerized on the surface of the shell layer.
Drawings
FIG. 1 is a flow chart of a method for preparing a quantum dot fluorescent probe according to an embodiment of the present invention;
FIG. 2 is a diagram of an ultraviolet absorption spectrum of gold nanoparticles provided in example 1 of the present invention;
FIG. 3(a) is Au @ SiO provided in example 1 of the present invention2Transmission electron microscopy images of;
FIG. 3(b) is Au @ SiO provided in example 2 of the present invention2Transmission electron microscopy images of;
FIG. 3(c) is Au @ SiO provided in example 3 of the present invention2Transmission electron microscopy images of;
FIG. 3(d) is Au @ SiO provided in example 4 of the present invention2Transmission electron microscopy images of;
FIG. 3(e) is Au @ SiO provided in example 5 of the present invention2Transmission electron microscopy images of;
FIG. 4(a) is Au @ SiO solid provided in example 1 of the present invention2Transmission electron micrographs of @ PNIPAM-QDs;
FIG. 4(b) is Au @ SiO provided in example 2 of the present invention2Transmission electron micrographs of @ PNIPAM-QDs;
FIG. 4(c) is Au @ SiO provided in example 3 of the present invention2Transmission electron micrographs of @ PNIPAM-QDs;
FIG. 4(d) is Au @ SiO provided in example 4 of the present invention2Transmission electron micrographs of @ PNIPAM-QDs;
FIG. 4(e) is Au @ SiO provided in example 5 of the present invention2Transmission electron micrographs of @ PNIPAM-QDs;
FIG. 5 is Au @ SiO2A graph of the particle size of the @ PNIPAM-QDs fluorescent probe as the temperature changes;
FIG. 6 is Au @ SiO2Graph showing the change of fluorescence of @ PNIPAM-QDs fluorescent probe with the change of temperature.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
As shown in fig. 1, a method for preparing a temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe includes the following steps:
(1) growing a silicon dioxide shell layer on the surface of the gold nano-particle to form Au @ SiO2；
(2) Isopropyl acrylamide (NIPAM) monomer, Acrylic Acid (AA), Ammonium Persulfate (APS) and Au @ SiO2Mixing in SiO2Surface polymerization to form SiO2Coating the surface with an isopropyl acrylamide monomer to obtain Au @ SiO2@ PNIPAM microspheres;
(3) mixing Au @ SiO2The @ PNIPAM microsphere is placed in EDC (1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride) aqueous solution, and then the quantum dot modified by amino is added to generate amidation reaction, so that the quantum dot is successfully connected with Au @ SiO2The reaction is carried out for 6 to 10 hours at normal temperature on the surface of the @ PNIPAM microsphere to form Au @ SiO2@ PNIPAM-QDs fluorescent probes.
Further, the step (1) includes:
mixing gold nanoparticles, tetraethoxysilane, sodium hydroxide, ethanol and water according to the volume ratio of 2000: 15: 167: 2985: 16670-2。
Further, the step (1) further comprises:
and silicon dioxide shells with different thicknesses grow on the surfaces of the gold nanoparticles by adding tetraethoxysilane with different contents.
Further, the preparation of the gold nanoparticles comprises:
in chloroauric acid (HAuCl)4) Adding water, sodium citrate and sodium borohydride to form seed liquidAdding water, Cetyl Trimethyl Ammonium Bromide (CTAB) and ascorbic acid into chloroauric acid to form a growth solution, wherein the volume ratio of the chloroauric acid to the water to the sodium citrate to the sodium borohydride is as follows: 5: 190: 5: 6, the concentration of chloroauric acid is 10mmol/1, the concentration of sodium citrate is 2.94mg/ml, the concentration of sodium borohydride is 3.783mg/ml, and the volume ratio of chloroauric acid, water, cetyl trimethyl ammonium bromide and ascorbic acid is as follows: 6: 114: 96: 1.6, the concentration of chloroauric acid is 10mmol/1, the concentration of hexadecyl trimethyl ammonium bromide is 0.2mol/l, the concentration of ascorbic acid is 17.6mg/ml, and the seed solution is added into the growth solution to prepare the gold nano-particles.
Further, the step (1) further comprises:
at Au @ SiO2Adding a silane coupling agent, and fully reacting to obtain Au @ SiO with double bonds on the surface2. Silane coupling agent and Au @ SiO2The ratio of (A) to (B) was 2. mu.l: 3 mg. At this time, Au @ SiO2In ethanol, Au @ SiO2The ratio of ethanol to ethanol was 3 mg: 3 ml.
Further, the reaction temperature after mixing in the step (2) is 70-80 ℃, and the reaction time is 5-8 h. In the step (2), isopropyl acrylamide monomer, acrylic acid, ammonium persulfate and Au @ SiO2The mass ratio of (A) to (B) is as follows: 45: 4.5: 15: 3, and ultrapure water is also added during the reaction, and the mass of the ultrapure water is Au @ SiO 21000 times the mass of (c).
In the step (3), Au @ SiO2The volume ratio of the @ PNIPAM microsphere, the EDC and the quantum dots is 3000: 200: 1-3000: 200: 100, and preferably 3000: 200: 8.
Example 1
19ml of ultrapure water was put into a 25ml bottle, and 0.5ml of HAuCl was sequentially added thereto4Aqueous solution, 0.5ml of 2.94mg/ml aqueous sodium citrate solution, 0.6ml of 3.783mg/ml sodium borohydride ice water solution, magnetically stirring at 30 ℃ for 5 minutes, and then standing for 3 hours to obtain a seed solution. 6.99744g CTAB was weighed into a 250ml Erlenmeyer flask, 210ml ultrapure water was added, magnetic stirring was carried out at 30 ℃ until dissolved, and then 6ml HAuCl was added4Aqueous solution, 1.6ml of 17.6mg/ml ascorbic acid aqueous solution, and the above seed solution were added, stirred for 1 hour, and left to standFor 12 hours. Taking 32ml of the aqueous solution of the synthesized gold nanoparticles, centrifuging the aqueous solution at 15000rpm for 30 minutes, removing the supernatant, adding 16.67ml of ultrapure water, adding 167 mu l of 0.1mol/ml aqueous solution of sodium hydroxide, magnetically stirring the aqueous solution at 30 ℃ for 20 minutes to prepare three parts of an ethanol solution of tetraethyl orthosilicate, wherein 5 mu l of tetraethyl orthosilicate and 995 mu l of ethanol are added in one part per hour, reacting the three parts for 24 hours after the addition is finished, centrifuging the solution at 11000rpm for 20 minutes, washing the solution twice with water and washing the solution twice with ethanol, and obtaining Au @ SiO2Dispersed in ethanol. To obtain Au @ SiO2Adding 2 mul of silane coupling agent into the solution, magnetically stirring for 5 hours at 30 ℃, centrifuging for 20 minutes at 11000rpm, washing with ethanol twice and washing with water twice to obtain Au @ SiO with double bonds on the surface2. Weighing 45mg of isopropyl acrylamide, 4.5mg of acrylic acid, 15mg of ammonium persulfate and 3ml of ultrapure water, and mixing the Au @ SiO with double bonds on the surface obtained in the previous step2Adding the mixture into the solution, magnetically stirring the mixture for 5 hours at the temperature of 80 ℃, cooling the mixture to room temperature, and centrifuging the cooled mixture to obtain Au @ SiO with carboxyl on the surface2@ PNIPAM microstructure. Dispersing the product in an aqueous solution, adding 200 mu 120 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, then adding amino modified quantum dots, stirring at room temperature for 6 hours, centrifuging at 11000rpm for 20 minutes, washing with water for 3 times to obtain Au @ SiO2The structure of @ PNIPAM-QDs.
Example 2
19ml of ultrapure water was put into a 25ml bottle, and 0.5ml of HAuCl was sequentially added thereto4Aqueous solution, 0.5ml of 2.94mg/ml aqueous sodium citrate solution, 0.6ml of 3.783mg/ml sodium borohydride ice water solution, magnetically stirring at 30 ℃ for 5 minutes, and then standing for 3 hours to obtain seed liquid. 6.99744g CTAB was weighed into a 250ml Erlenmeyer flask, 210ml ultrapure water was added, magnetic stirring was carried out at 30 ℃ until dissolved, and then 6ml HAuCl was added4Aqueous solution, 1.6ml of aqueous solution of 17.6mg/ml ascorbic acid, and the above seed solution were added, stirred for 1 hour, and left to stand for 12 hours. 32ml of the aqueous solution of the synthesized gold nanoparticles was centrifuged at 15000rpm for 30 minutes, the supernatant was removed, 16.67ml of ultrapure water was added, and 167. mu.l of 0.1mol/ml sodium hydroxide water was addedMagnetically stirring the solution at 30 ℃ for 20 minutes to prepare three parts of ethanol solution of tetraethyl orthosilicate, wherein 10 mu l of tetraethyl orthosilicate and 990 mu l of ethanol are added one part per hour, reacting for 24 hours after the three parts are added, centrifuging at 11000rpm for 20 minutes, washing twice with water and twice with ethanol, and obtaining Au @ SiO2Dispersed in ethanol. To obtain Au @ SiO2Adding 2 mul of silane coupling agent into the solution, magnetically stirring for 5 hours at 30 ℃, centrifuging for 20 minutes at 11000rpm, washing with ethanol twice and washing with water twice to obtain Au @ SiO with double bonds on the surface2. Weighing 45mg of isopropyl acrylamide, 4.5mg of acrylic acid, 15mg of ammonium persulfate and 3ml of ultrapure water, and mixing the Au @ SiO with double bonds on the surface obtained in the previous step2Adding the mixture into the solution, magnetically stirring the mixture for 8 hours at 70 ℃, cooling the mixture to room temperature, and centrifuging the cooled mixture to obtain Au @ SiO with carboxyl on the surface2@ PNIPAM microstructure. Dispersing the product in an aqueous solution, adding 200 mu l of 20mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, then adding amino modified quantum dots, stirring for 6 hours at room temperature, centrifuging at 11000rpm for 20 minutes, washing for 3 times to obtain Au @ SiO2The structure of @ PNIPAM-QDs.
Example 3
19ml of ultrapure water was put into a 25ml bottle, and 0.5ml of HAuCl was sequentially added thereto4Aqueous solution, 0.5ml of 2.94mg/ml aqueous sodium citrate solution, 0.6ml of 3.783mg/ml sodium borohydride ice water solution, magnetically stirring at 30 ℃ for 5 minutes, and then standing for 3 hours to obtain seed liquid. 6.99744g CTAB was weighed into a 250ml Erlenmeyer flask, 210ml ultrapure water was added, magnetic stirring was carried out at 30 ℃ until dissolved, and then 6ml HAuCl was added4Aqueous solution, 1.6ml of aqueous solution of 17.6mg/ml ascorbic acid, and the above seed solution were added, stirred for 1 hour, and left to stand for 12 hours. Taking 32ml of the water solution of the synthesized gold nanoparticles, centrifuging the gold nanoparticles for 30 minutes at 15000rpm, removing supernatant, adding 16.67ml of ultrapure water, adding 167 mu l of 0.1mol/ml sodium hydroxide water solution, magnetically stirring the mixture for 20 minutes at 29 ℃, preparing three parts of ethanol solution of tetraethyl orthosilicate, wherein 15 mu l of tetraethyl orthosilicate and 985 mu l of ethanol are added in one part per hour, reacting the mixture for 26 hours after the three parts are added, centrifuging the mixture for 20 minutes at 11000rpm, washing the mixture twice with water and washing the mixture twice with ethanol to obtain the gold nanoparticlesTo Au @ SiO2Dispersed in ethanol. To obtain Au @ SiO2Adding 2 mul of silane coupling agent into the solution, magnetically stirring for 5 hours at 30 ℃, centrifuging for 20 minutes at 11000rpm, washing with ethanol twice and washing with water twice to obtain Au @ SiO with double bonds on the surface2. Weighing 45mg of isopropyl acrylamide, 4.5mg of acrylic acid, 15mg of ammonium persulfate and 3ml of ultrapure water, and mixing the Au @ SiO with double bonds on the surface obtained in the previous step2Adding the mixture into the solution, magnetically stirring the mixture for 5 hours at the temperature of 80 ℃, cooling the mixture to room temperature, and centrifuging the cooled mixture to obtain Au @ SiO with carboxyl on the surface2@ PNIPAM microstructure. Dispersing the product in an aqueous solution, adding 200 mu l of 20mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, then adding amino modified quantum dots, stirring for 6 hours at room temperature, centrifuging at 11000rpm for 20 minutes, washing for 3 times to obtain Au @ SiO2The structure of @ PNIPAM-QDs.
Example 4
19ml of ultrapure water was put into a 25ml bottle, and 0.5ml of HAuCl was sequentially added thereto4Aqueous solution, 0.5ml of 2.94mg/ml aqueous sodium citrate solution, 0.6ml of 3.783mg/ml sodium borohydride ice water solution, magnetically stirring at 30 ℃ for 5 minutes, and then standing for 3 hours to obtain seed liquid. 6.99744g CTAB was weighed into a 250ml Erlenmeyer flask, 210ml ultrapure water was added, magnetic stirring was carried out at 30 ℃ until dissolved, and 6ml HAuCl was added4Aqueous solution, 1.6ml of aqueous solution of 17.6mg/ml ascorbic acid, and the above seed solution were added, stirred for 1 hour, and left to stand for 12 hours. Taking 32ml of the aqueous solution of the synthesized gold nanoparticles, centrifuging the aqueous solution at 15000rpm for 30 minutes, removing supernatant, adding 16.67ml of ultrapure water, adding 167 mu l of 0.1mol/ml aqueous solution of sodium hydroxide, magnetically stirring the aqueous solution at 30 ℃ for 20 minutes to prepare three parts of ethanol solution of tetraethyl orthosilicate, wherein 20 mu l of tetraethyl orthosilicate and 980 mu l of ethanol are added, one part of tetraethyl orthosilicate is added every hour, reacting the mixture for 24 hours after the three parts are added, centrifuging the mixture at 11000rpm for 20 minutes, washing the mixture twice and washing the mixture with ethanol, and obtaining Au @ SiO2Dispersed in ethanol. To obtain Au @ SiO2Adding 2 mul of silane coupling agent into the solution, magnetically stirring for 5 hours at 30 ℃, centrifuging for 20 minutes at 11000rpm, washing with ethanol twice and washing with water twice to obtain Au @ SiO with double bonds on the surface2. Weighing 45mg of isopropylacrylamide, 4.5mg of acrylic acid, 15mg of ammonium persulfate and 3ml of ultrapure water, and reacting the Au @ SiO with double bonds on the surface obtained in the previous step2Adding the mixture into the solution, magnetically stirring the mixture for 5 hours at the temperature of 80 ℃, cooling the mixture to room temperature, and centrifuging the cooled mixture to obtain Au @ SiO with carboxyl on the surface2@ PNIPAM microstructure. Dispersing the product in an aqueous solution, adding 200 mu l of 20mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, then adding amino modified quantum dots, stirring at room temperature for 8 hours, centrifuging at 11000rpm for 20 minutes, washing with water for 3 times to obtain Au @ SiO2The structure of @ PNIPAM-QDs.
Example 5
19ml of ultrapure water was put into a 25ml bottle, and 0.5ml of HAuCl was sequentially added thereto4Aqueous solution, 0.5ml of 2.94mg/ml aqueous sodium citrate solution, 0.6ml of 3.783mg/ml sodium borohydride ice water solution, magnetically stirring at 30 ℃ for 5 minutes, and then standing for 3 hours to obtain seed liquid. 6.99744g CTAB was weighed into a 250ml Erlenmeyer flask, 210ml ultrapure water was added, magnetic stirring was carried out at 30 ℃ until dissolved, and then 6ml HAuCl was added4Aqueous solution, 1.6ml of aqueous solution of 17.6mg/ml ascorbic acid, and the above seed solution were added, stirred for 1 hour, and left to stand for 12 hours. Taking 32ml of the aqueous solution of the synthesized gold nanoparticles, centrifuging the aqueous solution at 15000rpm for 30 minutes, removing supernatant, adding 16.67ml of ultrapure water, adding 167 mu l of 0.1mol/ml aqueous solution of sodium hydroxide, magnetically stirring the aqueous solution at 32 ℃ for 20 minutes to prepare three parts of an ethanol solution of tetraethyl orthosilicate, wherein 25 mu l of tetraethyl orthosilicate and 975 mu l of ethanol are added, one part is added every hour, the reaction is carried out for 22 hours after the three parts are added, centrifuging the aqueous solution at 11000rpm for 20 minutes, washing the solution twice with water and the solution twice with ethanol, and obtaining Au @ SiO2Dispersed in ethanol. To obtain Au @ SiO2Adding 2 mul of silane coupling agent into the solution, magnetically stirring for 5 hours at 30 ℃, centrifuging for 20 minutes at 11000rpm, washing with ethanol twice and washing with water twice to obtain Au @ SiO with double bonds on the surface2. Weighing 45mg of isopropyl acrylamide, 4.5mg of acrylic acid, 15mg of ammonium persulfate and 3ml of ultrapure water, and mixing the Au @ SiO with double bonds on the surface obtained in the previous step2Adding the mixture into the solution, magnetically stirring the mixture for 6 hours at the temperature of 75 ℃, cooling the mixture to room temperature, and centrifuging the cooled mixture to obtain Au @ SiO with carboxyl on the surface2@PNIPAM microstructure. Dispersing the product in an aqueous solution, adding 200 mul of 20mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride aqueous solution, then adding amino modified quantum dots, stirring for 10 hours at room temperature, centrifuging at 11000rpm for 20 minutes, and washing for 3 times to obtain Au @ SiO2The structure of @ PNIPAM-QDs.
FIG. 2 is a graph showing the ultraviolet absorption spectrum of gold nanoparticles synthesized in example 1, and it can be seen that the absorption position is about 525 nm.
FIGS. 3(a) -3(e) are Au @ SiO prepared in examples 1-52The difference between the transmission electron microscope images is that TEOS addition amount is different, and SiO obtained by using TEOS with different amount can be seen2The shell thickness also varies.
FIGS. 4(a) -4(e) are Au @ SiO prepared in examples 1-52In a transmission electron microscope picture of @ PNIPAM-QDs, it can be obviously seen that Quantum Dots (QDs) are successfully connected to Au @ SiO2@ PNIPAM microsphere surface.
FIG. 5 shows Au @ SiO prepared in examples 1-52Temperature-responsive particle size plots of @ PNIPAM-QDs measured using Dynamic Light Scattering (DLS) show that as temperature increases, the particle size of the microspheres decreases, consistent with the theory of temperature-increasing shrinkage of PNIPAM.
FIG. 6 is a representation of Au @ SiO prepared in examples 1-52The fluorescence spectrum of temperature response of @ PNIPAM-QDs, measured with a fluorescence spectrophotometer, shows that the fluorescence decreases with increasing temperature because the PNIPAM temperature increases and shrinks, so that the distance between the quantum dot and gold approaches, and the quenching increases.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. The preparation method of the temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe is characterized in that the fluorescent probe is Au @ SiO applied to temperature measurement2@PNIPAM-QDsThe preparation method of the fluorescent probe comprises the following steps:
(1) growing a silicon dioxide shell layer on the surface of the gold nano-particle to form Au @ SiO2；
(2) Isopropyl acrylamide monomer, acrylic acid, ammonium persulfate and Au @ SiO2Mixing in SiO2Surface polymerization to form SiO2Coating the surface with an isopropyl acrylamide monomer to obtain Au @ SiO2@ PNIPAM microspheres;
(3) mixing Au @ SiO2The @ PNIPAM microsphere is placed in EDC aqueous solution, and then the quantum dot modified by amino is added to carry out amidation reaction, so that the quantum dot is successfully connected with Au @ SiO2Forming Au @ SiO on the surface of the @ PNIPAM microsphere2@ PNIPAM-QDs fluorescent probes.
2. The method for preparing a quantum dot fluorescent probe according to claim 1, wherein the step (1) comprises:
mixing gold nanoparticles, ethyl orthosilicate, sodium hydroxide and ethanol solution, and reacting at 29-32 ℃ for 22-26 h to grow a silicon dioxide shell layer on the surface of the gold nanoparticles to form Au @ SiO2。
3. The method for preparing a quantum dot fluorescent probe according to claim 2, wherein the step (1) further comprises:
and silicon dioxide shells with different thicknesses grow on the surfaces of the gold nanoparticles by adding tetraethoxysilane with different contents.
4. The method for preparing a quantum dot fluorescent probe according to any one of claims 1 to 3, wherein the preparation of the gold nanoparticles comprises:
adding water, sodium citrate and sodium borohydride into chloroauric acid to form seed liquid, adding water, cetyl trimethyl ammonium bromide and ascorbic acid into chloroauric acid to form growth liquid, and adding the seed liquid into the growth liquid to prepare gold nanoparticles.
5. The method for preparing a quantum dot fluorescent probe according to any one of claims 1 to 3, wherein the step (1) further comprises:
at Au @ SiO2Adding a silane coupling agent, and fully reacting to obtain Au @ SiO with double bonds on the surface2。
6. The method for preparing a quantum dot fluorescent probe according to any one of claims 1 to 3, wherein the reaction temperature after mixing in the step (2) is 70 ℃ to 80 ℃, and the reaction time is 5h to 8 h.
7. The method for preparing a quantum dot fluorescent probe according to any one of claims 1 to 3, wherein the particle size of the gold nanoparticles is 1nm to 100 nm.
8. The method for preparing a quantum dot fluorescent probe according to any one of claims 1 to 3, wherein the thickness of the silica shell layer is 2nm to 50 nm.
9. A temperature-responsive polymer grafted gold nanoparticle composite quantum dot fluorescent probe, which is characterized in that the quantum dot fluorescent probe is prepared by the preparation method of the quantum dot fluorescent probe as claimed in any one of claims 1 to 8.
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