CN114214391A - Au/Fe3O4Composite magnetic nano motor material, preparation method and application - Google Patents
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Abstract
The invention discloses Au/Fe3O4Composite magnetic nano motor material, preparation method and application thereofThe application comprises the following steps: firstly, preparing gold nanoparticles by a reduction method, mixing the gold nanoparticles with PAA to prepare Au/PAA JNTPs, and synthesizing Au/Fe (OH) by a TEOS reagent3‑PAA)@SiO2NPs, and finally preparing Au/Fe by a high-temperature oil-thermal method3O4@ C JNPs magnetic nano motor material. The magnetic nano motor can accurately control the direction movement according to the magnetic field, and has better effect on the circular capture and hybridization of genes; the accuracy of early diagnosis of the breast cancer is improved by realizing rapid, sensitive and direct detection of HER-2/neu, BRCA1 and BRCA2 genes of the breast cancer. The superparamagnetism of the magnetic nano motor can efficiently enrich the free nucleic acid in a serum sample to be detected, and is expected to realize the detection of whole blood.
Description
Technical Field
The invention relates to Au/Fe3O4A composite magnetic nano motor material, a preparation method and an application belong to the technical field of medicines.
Background
The HER-2/neu, BRCA1 and BRCA2 genes have important functions on the occurrence, development, diagnosis and treatment of breast cancer. HER-2/neu is called human epidermal growth factor receptor 2 (HER-2), and a large number of studies show that the incidence rate of HER-2/neu gene overexpression in female breast simple hyperplasia, atypical hyperplasia and breast cancer is about 0%, 22.5% and 46.7%, wherein the incidence rate in primary invasive breast cancer is 22% -29%, and the incidence rate in acne type ductal carcinoma in situ is almost 100%. Therefore, the HER-2/neu gene protein has important clinical value for early diagnosis of female breast cancer. BRCA1 genomic DNA is approximately 80kb long, and BECA2 genomic DNA is approximately 70kb long. The detection of mutations in BRCA1 and BRCA2 genes in women with family history of breast cancer shows that about 10% of such people are carriers of mutations in BRCA1 and BRCA2 genes. Further research shows that the risk of the breast cancer of carriers with BRCA1 and BRCA2 gene mutation is accumulated to 80-85% by age 70, and the risk of the breast cancer in life can reach 90%. Therefore, the detection of HER-2/neu, BRCA1 and BRCA2 genes is an effective method for early screening and early diagnosis of breast cancer.
The nanometer motor is a nanometer scale power machine, is a lead of nanometer world mechanization, has a machine working cycle, and is a machine system in a strict sense. The nano-scale micro-nano targeted drug delivery system can be directly applied to nano-targeted delivery of precisely and directionally delivered drugs, and lays a foundation for advanced mechanical devices and energy conversion devices with micro-nano sizes. Brownian motion is the automatic movement capability of particles generated by random colloidal motion, and the motion mechanism of nanomotors is mainly the result of conversion of chemical energy or external energy into mechanical motion, so that nanomotor technology can provide efficient fluid mixing. Numerous studies have shown that this technique has been used to capture, pick up and transport target substances. Compared with other materials, the nano motor has more freedom in selection of driving and controlling means.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides Au/Fe3O4A composite magnetic nano motor material, a preparation method and application.
In order to achieve the purpose, the invention adopts Au/Fe3O4The preparation method of the composite magnetic nano motor material comprises the following steps: firstly, preparing gold nanoparticles by a reduction method, mixing the gold nanoparticles with PAA to prepare Au/PAA JNTPs, and synthesizing Au/Fe (OH) by a TEOS reagent3-PAA)@SiO2NPs, and finally preparing Au/Fe by a high-temperature oil-thermal method3O4@ C JNPs magnetic nano motor material.
Preferably, the preparation method specifically comprises the following steps:
(1) synthesis of Au/PAA JNPs: reducing tetrachloroauric acid with sodium citrate to prepare monodisperse gold nanoparticles, washing the synthesized suspension of 100mL Au NPs with water to remove unreacted substances, redispersing the Au NPs in 24-26 mL deionized water, and then adding 50-100 mu L of 0.1-0.5 g/mL polyacrylic acid aqueous solution and 100-150 mu L of 2mol/L NH in a 250mL flask3·H2O, ultrasonically dispersing for 9-11 min, and adding 150-250 mL of isopropanol under magnetic stirring to obtain Au/PAA JNPs;
(2)Au/Fe(OH)3-synthesis of PAA JNPs: under strong magnetic stirring, 44-46 mu L of FeCl with the concentration of 0.3-0.6 g/mL2·4H2Adding the O aqueous solution into the synthesized 39-40 mL suspension of Au/PAA JNPsIn the method, a reaction mixture is stirred for 5-7 h, products are collected through centrifugation, JNPs obtained are purified through multiple centrifugation/redispersion in 9-11 mL of water, and 39-40 mL of isopropanol is added into Au/Fe (OH)3PAA JNPs, wherein the volume ratio of water to isopropanol is 1: 2-1: 5;
(3)(Janus Au/Fe(OH)3-PAA)@SiO2synthesis of NPs: mixing Au/Fe (OH)3Adjusting the pH of the suspension of-PAA JNPs to 7-8, adding 9-11 mu L of tetraethoxysilane with purity of more than or equal to 98% into the suspension, and stirring at room temperature for 23-25 h to obtain (Janus Au/Fe (OH)3-PAA)@SiO2NPs are generated through centrifugal collection, are repeatedly washed by deionized water to remove redundant precursors, and then are placed in a vacuum drying chamber for 11-13 h;
(4) monodisperse Au/Fe3O4Synthesis of @ C JNPs: will (Janus Au/Fe (OH)3-PAA)@SiO2NPs are heated to 400-500 ℃ at the speed of 2 ℃/min under high-purity argon, and are maintained at the temperature for 3-5 h to obtain monodispersion (Janus Au/Fe)3O4@C)@SiO2NPs, dispersing the obtained JNPs in 3-5 mL of water, then etching with 200mL of 2mol/L sodium hydroxide aqueous solution at 50 ℃, centrifuging and collecting Au/Fe after 23-25 h3O4@ C JNPs, washed with IPA and water.
The invention also provides Au/Fe prepared by the preparation method3O4Composite magnetic nanomotor materials.
In addition, the invention also provides the Au/Fe3O4The application of the composite magnetic nano motor material in preparing a gene detection probe for early screening of breast cancer.
Preferably, the gene detection probe is used for combined capture and detection of HER-2/neu, BRCA1 and BRCA2 genes.
As another preferred aspect of the present invention, the base sequence of the gene assaying probe is complementarily paired with a base sequence of a part of the target gene;
the method comprises the following specific steps: after the magnetic nano motor material is washed, 1mL of HS-DNA-Cy3 with the concentration of 1-5 mu mol/L is added to 1mg/mL of Au/Fe3O4In water suspension of @ C JNPs, the mixture is subjected to ultrasonic treatment for 30 seconds and is kept stand for reaction overnight; centrifugally collecting the obtained DNA-Au/Fe3O4@ C JNPs, washed with PBS to remove excess HS-DNA to obtain DNA-Au/Fe3O4@C JNPs。
Finally, the invention also provides a catalyst containing the Au/Fe3O4A gene detection probe of the composite magnetic nano motor material.
The principle of the magnetic nano motor for capturing the breast cancer target gene is as follows: firstly, preparing prepared Au/Fe3O4The @ CJNPs magnetic nano-motor material is combined with a Cy3 modified thiolated DNA molecular fragment (HS-DNA-Cy3) probe to prepare a high-specificity magnetic nano-motor detection probe. Under the condition that a target object exists, the probe can efficiently and specifically enrich the target gene in a sample to be detected from a clinical sample, the target gene is combined on the probe to play a role in capturing nucleic acid, and the Cy3 fluorescence is recovered. When the target gene is not present, the probe cannot bind to the target substance from the sample, and therefore, the subsequent reaction cannot proceed, the Cy3 fluorescence detection signal is not generated, the detection result specificity is good, and the Cy3 fluorescence detection signal is not formed by binding to a non-target gene.
Compared with the prior art, the invention has the beneficial effects that:
(1) compared with the magnetic nano-microspheres on the market, the magnetic nano-microsphere has better effect. The magnetic nano motor can accurately control the direction movement according to the magnetic field, and has better effect on the circular capture and hybridization of genes.
(2) The accuracy of early diagnosis of the breast cancer is improved by realizing rapid, sensitive and direct detection of HER-2/neu, BRCA1 and BRCA2 genes of the breast cancer.
(3) The superparamagnetism of the magnetic nano motor can efficiently enrich the free nucleic acid in a serum sample to be detected, and is expected to realize the detection of whole blood.
Drawings
FIG. 1 is a process for preparing a magnetic nanomotor detection probe of the present invention;
FIG. 2 is an electron microscope representation of the magnetic nanomotor of the present invention;
FIG. 3 is a diagram showing the effect of the magnetic nanomotor of the present invention on the capture of target nucleic acid by hybridization.
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. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention.
Referring to fig. 1, the implementation steps of the present invention are as follows:
(1) preparing a magnetic nano motor material by using a reducing agent high-temperature hydrothermal method, accurately weighing the magnetic nano motor material, transferring a certain amount of HS-DNA-Cy3 by using a liquid transfer gun, dissolving in a PBS reaction solution, mixing the solution in an EP tube, placing the EP tube on a rotary incubator, regulating the rotating speed of the rotary incubator, and standing overnight for reaction; and then transferring the liquid into a pointed EP tube, carrying out magnetic separation and washing, fully collecting precipitates, washing to obtain a magnetic nano motor detection probe, and fixing the detection probe in a 96-well detection plate.
(2) The primers and DNA sequences used are all provided by Shanghai's biosynthesized.
Example 1
Designing the specific HS-DNA-Cy3 of the magnetic nano motor for capturing the nucleic acid gene: by analyzing the sequence of the target gene, a specific HS-DNA-Cy3 probe is selected and designed according to the difference between the paired Tm temperature value and the delta G energy value.
The Tm temperature values of the target gene pair are 58.2 ℃, 49.7 ℃ and 53.2 ℃, the energy values of delta G are 1.18kcal/mole, -1.50kcal/mole and 1.2kcal/mole respectively, so the Tm value of the designed hDNA probe is higher than 58.2 ℃, 49.7 ℃, 53.2 ℃ and the energy values of delta G are lower than 1.18kcal/mole, -1.50kcal/mole and 1.2kcal/mole, and the hDNA probe prepared in the way can efficiently and specifically combine with the target gene. The specificity of hDNA was evaluated and hDNA with the best specificity was selected. The nucleic acid sequences used therein are shown in Table 1 below.
TABLE 1 nucleic acid sequences
Example 2
Au/Fe3O4The preparation method of the composite magnetic nano motor material specifically comprises the following steps:
(1) monodisperse gold nanoparticles having a particle size of about 50nm were prepared by reducing tetrachloroauric acid with sodium citrate, a suspension of the synthesized Au NPs (100mL) was washed with water to remove unreacted materials and redispersed in 25mL of deionized water, and then 100. mu.L of polyacrylic acid (PAA) aqueous solution (0.2g/mL) and 125. mu.L of NH were added to a 250mL flask3·.H2O (2mol/L), ultrasonically dispersing for 10min, and adding 250mL of Isopropanol (IPA) under magnetic stirring to obtain Au/PAA JNPs;
(2) under vigorous magnetic stirring, 45. mu.L of FeCl with a concentration of 0.5g/mL2·4H2Adding aqueous O solution to a 40mL suspension of synthesized Au/PAA JNPs, stirring the reaction mixture for 6h, centrifuging to collect the product, obtaining JNPs, purifying by 3 centrifugation/redispersion in 10mL water, adding 40mL IPA to Au/Fe (OH)3Further experiments were performed in PAA JNPs (water/IPA volume ratio 1: 4);
(3) with 50mL of NH3·H2O solution mixing Au/Fe (OH)3The suspension of-PAA JNPs is adjusted to pH 8, Tetraethoxysilane (TEOS) (≧ 98%; 10 μ L) is added to the suspension, stirring is carried out at room temperature for 24h, obtaining (Janus Au/Fe (OH)3-PAA)@SiO2NPs, centrifugally collecting generated NPs, repeatedly washing with deionized water to remove redundant precursors, and then putting the NPs into a vacuum drying chamber for 12 h;
(4)(Janus Au/Fe(OH)3-PAA)@SiO2NPs were heated to about 500 ℃ at a rate of 2 ℃/min under high purity argon and maintained at this temperature for 4 hours to obtain monodispersion (Janus Au/Fe)3O4@C)@SiO2NPs; the obtained JNPs were dispersed in water (4mL) and then etched with 200mL of a 2mol/L aqueous solution of sodium hydroxide at 50 ℃. After 24h, the Au/Fe was collected by centrifugation3O4@ C JNPs, washed with IPA and water. At Au/Fe3O4In the expression of @ C JNPs, the symbol "@" denotes Fe3O4Part of which is encapsulated in carbon, while the Au component is exposed.
An electron microscope characterization diagram of the magnetic nano motor prepared in the embodiment 2 is shown in fig. 2, and analysis of fig. 2 shows that the magnetic nano motor prepared through experiments has good properties and meets expected requirements.
Example 3
Au/Fe3O4The preparation method of the composite magnetic nano motor material specifically comprises the following steps:
(1) monodisperse gold nanoparticles having a particle size of about 50nm were prepared by reducing tetrachloroauric acid with sodium citrate, a suspension of the synthesized Au NPs (100mL) was washed with water to remove unreacted materials and redispersed in 24mL of deionized water, and then 50. mu.L of an aqueous PAA solution (0.1g/mL) and 100. mu.L of NH were added to a 250mL flask3·H2O (2mol/L), ultrasonically dispersing for 9min, and adding 150mL of IPA under magnetic stirring to obtain Au/PAA JNPs;
(2) 44 μ L FeCl at a concentration of 0.3g/mL under vigorous magnetic stirring2·4H2Adding aqueous O solution to a suspension of synthesized 39mL Au/PAA JNPs, stirring the reaction mixture for 5h, centrifuging to collect the product, obtaining JNPs, purifying by 3 centrifugation/redispersion in 9mL water, adding 39mL IPA to Au/Fe (OH)3Further experiments were performed in PAA JNPs (water/IPA volume ratio 1: 2);
(3) with 49mL of NH3·H2O solution mixing Au/Fe (OH)3Adjusting the pH value of the suspension of-PAA JNPs to be approximately equal to 7, adding TEOS (more than or equal to 98%; 9 mu L) into the suspension, and stirring at room temperature for 23h to obtain (Janus Au/Fe (OH))3-PAA)@SiO2NPs, which are generated by collecting NPs through centrifugation and repeatedly washed with deionized water to remove excess precursors, are then placed in a vacuum drying chamber for 11 h;
(4)(Janus Au/Fe(OH)3-PAA)@SiO2NPs were heated to about 400 ℃ at a rate of 2 ℃/min under high purity argon and maintained at this temperature for 3 hours to obtain monodispersion (Janus Au/Fe)3O4@C)@SiO2NPs, the JNPs obtained were dispersed in water (3mL) and then etched with 200mL of 2mol/L aqueous sodium hydroxide solution at 50 ℃ for 23h, and then collected by centrifugation3O4@ C JNPs, washed with IPA and water.
Example 4
Magnetic nano motor detection probe DNA-Au/Fe3O4The preparation method of the @ C JNPs comprises the following steps:
washing 1mg of magnetic nanomotor with 0.01mol/L Phosphate Buffer Solution (PBS) for three times, and then adding a certain volume of HS-DNA-Cy3 (activated by tris (2-carboxyethyl) phosphine with the concentration of 1 mg/mL) with the concentration of 1 mu mol/L, wherein the magnetic nanomotor material and the HS-DNA-Cy3 are stood in 500 mu L of 0.01mol/L PBS for overnight reaction according to the mass molar ratio of 1mg (500 pmol-1000 pmol); after the reaction is finished, separating and purifying by adopting a magnetic frame, washing at least three times by using 0.01mol/L PBS (PBST) containing 0.2% -2% of Tween, removing unreacted HS-DNA-Cy3, and obtaining the high-specificity DNA-Au/Fe labeled by HS-DNA-Cy33O4@ C JNTPs magnetic nanomotor probes.
Example 5
DNA-Au/Fe3O4A method for capturing breast cancer related genes by a @ C JNTPs magnetic nano motor comprises the following steps:
high-specificity magnetic nano motor probe DNA-Au/Fe3O4And (2) adding the @ C JNPs into a 96-hole detection plate, mixing a target gene to be detected and a probe into PBS according to the volume ratio of 1: 5-1: 1, placing the mixture into an incubator, reacting at 37-50 ℃ for 20-45 min, and then carrying out magnetic separation and purification to remove supernatant, wherein non-target gene molecules can be removed in the step. After the magnetic separation is finished, 10-25 mu L of PBS is added for resuspension, the supernatant is removed, and 0.0 is usedWashing the sample for 2-5 times by using 1mol/L PBST, carrying out magnetic separation and purification to remove a supernatant, adding 10-25 mu L PBS for resuspension, and reading the intensity value of a Cy3 fluorescence signal by using a multifunctional microplate reader.
Example 6
DNA-Au/Fe3O4Verification that the @ C JNTPs magnetic nano motor captures breast cancer related genes:
preparing a sample containing a target substance, and detecting a high-specificity magnetic nano motor probe DNA-Au/Fe3O4The @ C JNPs are added into a 96-hole detection plate, target genes to be detected and probes are mixed in PBS according to the volume ratio of 1:3, the mixture is placed in an incubator and reacts for 38min at 37 ℃, and then supernatant is removed by magnetic separation and purification, and non-target gene molecules are removed;
after the magnetic separation, 15 μ L of PBS was added for resuspension, the supernatant was removed, washed 4 times with 0.01mol/L PBST, the supernatant was removed by magnetic separation and purification, 15 μ L of PBS was added for resuspension, and the intensity of Cy3 fluorescence signal was read with a multifunctional microplate reader.
The verification result is shown in fig. 3, which proves that the probe can efficiently and specifically enrich the target gene in the sample to be detected from the clinical sample in the presence of the target substance, the target gene is combined to the probe to play a role in nucleic acid capture, the Cy3 fluorescence is recovered to generate a Cy3 fluorescence detection signal, the detection result has good specificity, and the Cy3 fluorescence detection signal is not formed by combining with the non-target gene.
Example 7
In the method for capturing nucleic acid genes by using the magnetic nano motor, the addition amount of a magnetic nano motor probe is optimized: and taking a sample to be detected containing 10-11 mol/L of target genes by using a pipette, respectively adding 0, 1, 2, 3, 4, 5, 6 and 7 mu g of prepared probes, performing magnetic separation and washing after the probe reacts with the sample to be detected, and collecting Cy3 fluorescent signals to obtain the optimal addition amount of the magnetic nano motor probe.
The magnetic nano motor material prepared by the invention combines the HS-DNA-Cy3 complementary sequence segment matched with the breast cancer related gene to prepare and generate DNA-Au/Fe3O4The @ C JNPs are combined with the magnetic nanomotor detection probe of the functional nucleic acid group. The material size andthe magnetic nano motor material and the magnetic nano motor detection probe provided by the invention realize the combined detection of breast cancer marker genes HER-2/neu, BRCA1 and BRCA2 in a body fluid sample, and simultaneously, the superparamagnetism of the magnetic nano motor material is used for enriching the serum to be detected, so that the accuracy and the sensitivity of early diagnosis of breast cancer are improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
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Claims (7)
1. Au/Fe3O4The preparation method of the composite magnetic nano motor material is characterized by comprising the following steps of: firstly, preparing gold nanoparticles by a reduction method, mixing the gold nanoparticles with PAA to prepare Au/PAA JNTPs, and synthesizing Au/Fe (OH) by a TEOS reagent3-PAA)@SiO2NPs, and finally preparing Au/Fe by a high-temperature oil-thermal method3O4@ C JNPs magnetic nano motor material.
2. Au/Fe according to claim 13O4The preparation method of the composite magnetic nano motor material is characterized by comprising the following steps:
(1) synthesis of Au/PAA JNPs: reducing tetrachloroauric acid with sodium citrate to prepare monodisperse gold nanoparticles, washing the synthesized suspension of 100mL Au NPs with water to remove unreacted substances, redispersing in 24-26 mL deionized water, and then placing in a 250mL flaskAdding 50-100 mu L of 0.1-0.5 g/mL polyacrylic acid aqueous solution and 100-150 mu L of 2mol/L NH3·H2O, ultrasonically dispersing for 9-11 min, and adding 150-250 mL of isopropanol under magnetic stirring to obtain Au/PAA JNPs;
(2)Au/Fe(OH)3-synthesis of PAA JNPs: under strong magnetic stirring, 44-46 mu L of FeCl with the concentration of 0.3-0.6 g/mL2·4H2Adding an O aqueous solution into a synthesized suspension of 39-40 mL of Au/PAA JNPs, stirring the reaction mixture for 5-7 h, centrifuging to collect a product, performing multiple centrifugation/redispersion purification on the JNPs in 9-11 mL of water, adding 39-40 mL of isopropanol into Au/Fe (OH)3PAA JNPs, wherein the volume ratio of water to isopropanol is 1: 2-1: 5;
(3)(Janus Au/Fe(OH)3-PAA)@SiO2synthesis of NPs: mixing Au/Fe (OH)3Adjusting the pH of the suspension of-PAA JNPs to 7-8, adding 9-11 mu L of tetraethoxysilane with purity of more than or equal to 98% into the suspension, and stirring at room temperature for 23-25 h to obtain (Janus Au/Fe (OH)3-PAA)@SiO2NPs are generated through centrifugal collection, are repeatedly washed by deionized water to remove redundant precursors, and then are placed in a vacuum drying chamber for 11-13 h;
(4) monodisperse Au/Fe3O4Synthesis of @ C JNPs: will (Janus Au/Fe (OH)3-PAA)@SiO2NPs are heated to 400-500 ℃ at the speed of 2 ℃/min under high-purity argon, and are maintained at the temperature for 3-5 h to obtain monodispersion (Janus Au/Fe)3O4@C)@SiO2NPs, dispersing the obtained JNPs in 3-5 mL of water, then etching with 200mL of 2mol/L sodium hydroxide aqueous solution at 50 ℃, centrifuging and collecting Au/Fe after 23-25 h3O4@ C JNPs, washed with IPA and water.
3. Au/Fe prepared by the preparation method of any one of claims 1-23O4Composite magnetic nanomotor materials.
4. Au/Fe as claimed in any of claims 1 to 33O4Composite magnetic nano motor materialThe application of the material in preparing a gene detection probe for early screening of breast cancer.
5. The use of claim 4, wherein said gene detection probes are used for the combined capture and detection of the HER-2/neu, BRCA1 and BRCA2 genes.
6. The use according to claim 4, wherein the base sequence of the gene detection probe is complementarily paired with a base sequence of a part of a target gene;
the method comprises the following specific steps: after the magnetic nano motor material is washed, 1mL of HS-DNA-Cy3 with the concentration of 1-5 mu mol/L is added to 1mg/mL of Au/Fe3O4In water suspension of @ C JNPs, the mixture is subjected to ultrasonic treatment for 30 seconds and is kept stand for reaction overnight; centrifugally collecting the obtained DNA-Au/Fe3O4@ C JNPs, washed with PBS to remove excess HS-DNA to obtain DNA-Au/Fe3O4@C JNPs。
7. A composition comprising Au/Fe as defined in any one of claims 1 to 33O4A gene detection probe of the composite magnetic nano motor material.
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