CN115430410A - Anilinized modified magnetic bead compound and preparation method and application thereof - Google Patents
Anilinized modified magnetic bead compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a magnetic bead compound modified by phenylated iron, a preparation method and application thereof 2 @Fe 3 O 4 Magnetic beads; the magnetic bead compound modified by phenylamine has stronger superparamagnetism, can move directionally under the action of a magnetic field, and is compared with the magnetic bead compound modified by phenylamineNH prepared from gamma-aminopropyltriethoxysilane 2 @SiO 2 @Fe 3 O 4 The magnetic beads have better dispersibility in the dispersing agent and better adsorption capacity to DNA, and can successfully elute the DNA from the magnetic beads; the preparation method of the magnetic bead compound modified by phenylamine is simple, the reaction conditions are mild, the complexity of the preparation process is avoided, and the danger in the synthesis process is greatly reduced.
Description
Technical Field
The invention belongs to the technical application of magnetic materials, and particularly relates to an aniline modified magnetic bead compound and a preparation method and application thereof.
Background
Magnetic Nanoparticles (MNPs) are nano-sized magnetic materials, which may be called magnetic beads, and have the dual characteristics of magnetic particles and Nanoparticles (NPs), such as magnetic guidance, biocompatibility, small size effect, surface effect, and certain biomedical functions. The small size effect makes the magnetic nanometer material appear black in nanometer state, the melting point is reduced, and certain superparamagnetism is provided. Meanwhile, the surface atom number of the magnetic beads is increased due to the surface effect of the nano particles, and the magnetic beads are easily combined with other atoms to be stabilized, so that the magnetic beads have high chemical activity. Besides the characteristics of the nano particles, the magnetic beads have certain magnetic conductivity, namely, the magnetic beads can move directionally under the guidance of a certain external magnetic field.
The exposed magnetic beads have larger specific surface energy, high magnetization intensity and high chemical activity, and the particles directly contacted with the air are very sensitive to oxygen and are easily oxidized due to the activity. And the particles have interaction, so that the magnetic beads are easy to agglomerate. Therefore, the magnetic beads need to be modified to some extent. New active functional groups such as hydroxyl, carboxylic acid, amino and the like are added on the surfaces of the magnetic particles in a copolymerization mode and the like, and due to the electrostatic effect, repulsive force exists among the magnetic particles, so that the probability of occurrence of oxidation and agglomeration is reduced. Meanwhile, due to the existence of the modifying group, the functionality of the magnetic bead is effectively improved. By adding or modifying functional groups attached to the particles, the coordination between the particles and organisms is improved, so that the particles are more suitable for various fields. At present, magnetic solid-phase extraction technology taking magnetic beads as cores is widely applied to various fields such as compound detection, food, pesticides, protein and polypeptide.
Currently, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane and the like are commonly used as amino-modified magnetic beads for DNA extraction, the reagents are single, some of the reagents even need to use a plurality of organic solvents with high toxicity such as acetone, toluene and the like, or strict reaction conditions such as heating or vacuum and the like are needed, and the adsorption capacity of the reagents on DNA is too strong due to steric hindrance of the 3-aminopropyltriethoxysilane, so that the reagents are difficult to elute from the magnetic beads.
The present invention has been made in view of the above circumstances.
Disclosure of Invention
The invention provides an aniline modified magnetic bead compound and a preparation method and application thereof, aiming at solving the problems that in the prior art, an aminosilane coupling agent used for performing amination modification on the surface of a magnetic bead is single in type, strict in reaction condition, complex in preparation process, too low in elution rate caused by too strong adsorption capacity of DNA on the magnetic bead and the like.
The invention provides a magnetic bead compound modified by aniline, which sequentially comprises a ferroferric oxide core layer, a silicon dioxide modification layer and an aniline group modification layer from inside to outside.
The second objective of the present invention is to provide a method for preparing the magnetic bead complex modified by aniline, wherein the method comprises the following steps:
(1) In FeCl 2 ·4H 2 O and FeCl 3 ·6H 2 Dripping a precipitator into the O mixed aqueous solution, washing and drying to obtain ferroferric oxide;
(2) Mixing the ferroferric oxide with ethanol, performing ultrasonic dispersion, adding a mixed solution of ethanol, ethanolamine and water, uniformly mixing, dropwise adding tetraethoxysilane under the condition of stirring, washing, and drying to obtain magnetic beads coated with silicon dioxide;
(3) Mixing the magnetic beads coated with the silicon dioxide with ethanol, performing ultrasonic dispersion, dropwise adding aniline methyl triethoxysilane under the stirring condition, washing, and drying to obtain the magnetic bead composite modified by aniline.
Further, feCl in the step (1) 2 ·4H 2 The concentration of O in the mixed aqueous solution is 0.146-0.150mol/L, feCl 3 ·6H 2 The concentration of O in the mixed water solution is 0.28-0.32mol/L, and the precipitator is NaOH solution with the mass fraction of 28-32%.
Furthermore, in the step (1), the drying temperature is-80 to-72 ℃, and the freeze-drying time is 22 to 26 hours.
Further, in the step (2), the mass-to-volume ratio of the ferroferric oxide to the ethanol is 25g to 2L to 4L, the volume ratio of the ethanol to the ethanolamine to the water in the mixed solution of the ethanol to the ethanolamine to the water is 28 to 32, 58 to 5.26 to 5.30, and the mass-to-volume ratio of the ferroferric oxide to the mixed solution of the ethanol to the ethanolamine to the water is 250mg.
Further, the ultrasonic dispersion time in the step (2) is 7-10min, and the ultrasonic frequency is 35-45kHz.
Further, in the step (2), the mass-volume ratio of the ferroferric oxide to the tetraethoxysilane is 250mg, the stirring speed is 250-350rpm when the tetraethoxysilane is dripped, and after the dropwise addition is finished, the reaction is carried out for 5-7h at the stirring speed of 450-550 rpm.
Furthermore, the drying temperature in the step (2) is-78 to-74 ℃, and the drying time is 16-20h.
Further, in the step (3), the mass-to-volume ratio of the magnetic beads coated with silicon dioxide to ethanol is 100mg, and the mass-to-volume ratio of the magnetic beads coated with silicon dioxide to aniline methyl triethoxysilane is 100mg.
And (3) further, dropwise adding aniline methyl triethoxysilane in the step (3) at a stirring speed of 450-600rpm, keeping the stirring speed unchanged, and continuously stirring for reaction for 0.8-1.2h to obtain the magnetic bead composite modified by aniline.
Further, the ultrasonic dispersion time in the step (3) is 13-17min, and the ultrasonic frequency is 35-45kHz. Furthermore, the drying temperature in the step (3) is-78 to-74 ℃, and the drying time is 22-26h.
The third purpose of the invention is to provide an application of the magnetic bead complex modified by aniline in the extraction of free DNA outside cells.
In the process of preparing the magnetic bead compound modified by aniline, the amount of aniline methyl triethoxysilane added is different, the number of groups modified on the magnetic beads is different, and the extraction effect on DNA is different. Since the magnetic beads are modified by using the aniline methyl triethoxysilane for the first time, no relevant information and data are available for determining the amount of the aniline methyl triethoxysilane. Therefore, the optimal dosage of the aniline methyl triethoxysilane is finally determined by continuously adjusting the dosage of the aniline methyl triethoxysilane in the aniline modified magnetic bead composite during the preparation of the amino modified magnetic bead composite.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention takes ferroferric oxide with paramagnetism as a basic nuclear layer of magnetic beads, and takes silicon dioxide and anilino compound as a modification layer of the magnetic beads to obtain a multilayer modified Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads;
(2) The magnetic bead compound modified by phenylamine has stronger superparamagnetism, can move directionally under the action of a magnetic field, and is compared with NH prepared from gamma-aminopropyltriethoxysilane 2 @SiO 2 @Fe 3 O 4 The magnetic beads have better dispersibility in the dispersing agent and better adsorption capacity to DNA, and can successfully elute the DNA from the magnetic beads;
(3) The preparation method of the magnetic bead compound modified by phenylamine is simple, the reaction conditions are mild, the reaction can be carried out only under the normal temperature condition, heating is not needed, the complexity of the preparation process is avoided, and the danger in the synthesis process is greatly reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the structure of a magnetic bead complex modified by aniline according to the present invention;
FIG. 2 is Fe prepared in example 1 of the present invention 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 SEM pictures of three magnetic beads of magnetic beads;
FIG. 3 is Fe prepared in example 1 of the present invention 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 XRD patterns of three magnetic beads of magnetic beads;
FIG. 4 is Fe prepared in example 1 of the present invention 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 FI-IR diagrams of three magnetic beads of magnetic beads;
FIG. 5 is Fe prepared in example 1 of the present invention 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 X-ray photoelectron energy spectrograms of three magnetic beads of the magnetic bead;
FIG. 6 is Fe prepared in example 1 of the present invention 3 O 4 Magnetic beads, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 VSM curves of three magnetic beads of the magnetic bead;
FIG. 7 is Fe prepared in example 1 of the present invention 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Zeta potential curves of three magnetic beads of magnetic beads;
FIG. 8 Fe prepared in example 1 of the present invention 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Three magnetic bead pairsDNA adsorption rate and elution efficiency.
Reference numerals
1-ferroferric oxide nuclear layer, 2-silicon dioxide modification layer and 3-aniline modification layer.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
In the embodiment of the invention, feCl 2 ·4H 2 O (99%) was purchased from Dorbett reagent, feCl 3 ·6H 2 O (99%) and absolute ethanol were purchased from Woodcondan chemical reagent factory, tetraethoxysilane (TEOS, 99%) was purchased from Aladdin reagent (Shanghai), ethanolamine (99%) was purchased from Anhuizengl technology Co., ltd, naOH (99%) was purchased from Tianjin Oxypro chemical Co., ltd, and phenylaminomethyltriethoxysilane (ND-42, 98%) was purchased from Shanghai Tantake technology Co., ltd.
Fig. 1 is a schematic structural diagram of a magnetic bead composite modified by aniline, and the magnetic bead composite sequentially includes a ferroferric oxide core layer 1, a silica modification layer 2, and an aniline modification layer 3 from inside to outside.
Example 1
The preparation method of the magnetic bead complex modified by aniline concretely comprises the following steps:
(1)Fe 3 O 4 preparing magnetic beads:
0.74g (3.7 mmol) FeCl was weighed 2 ·4H 2 O and 2.03g (7.5 mmol) FeCl 3 ·6H 2 Dissolving O in 25mL deionized water, stirring at room temperature at 500rpm/min, dropwise adding 7mL NaOH solution with concentration of 30% w/v after the solid is completely dissolved to form black precipitate, stirring for 1-2 hr,obtaining black magnetic particles, washing the black magnetic particles with deionized water until the pH value is neutral, and freeze-drying the black magnetic particles for 24 hours at the temperature of minus 76 ℃ to obtain black granular solid Fe 3 O 4 Magnetic beads;
(2)SiO 2 @Fe 3 O 4 preparing magnetic beads:
taking 250mg of Fe 3 O 4 Placing the mixture into 30mL of absolute ethyl alcohol for ultrasonic treatment for 7min, wherein the ultrasonic frequency is 40kHz, adding 30mL of ethyl alcohol, 60mL of ethanolamine and 5.28g of ultrapure water into a beaker, and uniformly stirring. Mixing the above liquid with Fe 3 O 4 Uniformly mixing the particles, putting the mixture into a three-neck flask, dropwise adding 0.6mL of tetraethyl orthosilicate (TEOS) while stirring at the rotating speed of 300rpm, adjusting the rotating speed to 500rpm after dropwise adding, mechanically stirring for 6h, washing the mixture with ethanol and deionized water for three times after the reaction is finished, and freeze-drying the mixture for 18h at the temperature of-76 ℃ to obtain SiO 2 @Fe 3 O 4 Magnetic beads, i.e., silica-coated magnetic beads;
(3)Ph-NH@SiO 2 @Fe 3 O 4 preparing magnetic beads:
taking 100mg of SiO 2 @Fe 3 O 4 Placing in 40mL anhydrous ethanol, ultrasonic dispersing for 15min with ultrasonic frequency of 40kHz, adding 100 μ L aniline methyl triethoxysilane (ND-42), mechanically stirring for 1h, washing with ethanol and deionized water respectively for three times after reaction, freeze drying for 24h at-76 deg.C to obtain Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads, i.e., magnetic bead complexes modified by anilination.
The magnetic bead composite modified by aniline prepared in this embodiment sequentially includes a ferroferric oxide core layer 1, a silica modification layer 2, and an anilino modification layer 3 from inside to outside.
Example 2
The preparation method of the magnetic bead complex modified by aniline concretely comprises the following steps:
(1)Fe 3 O 4 preparing magnetic beads:
0.726g (3.65 mmol) FeCl was weighed 2 ·4H 2 O and 1.89g (7.0 mmol) FeCl 3 ·6H 2 O,Dissolving in 25mL deionized water, stirring at room temperature at 500rpm/min, dropwise adding 7mL NaOH solution with concentration of 28 w/v after the solid is completely dissolved to generate black precipitate, stirring for 1-2 hr to obtain black magnetic particles, washing the obtained black magnetic particles with deionized water until pH is neutral, and lyophilizing at-80 deg.C for 22 hr to obtain black granular solid Fe 3 O 4 Magnetic beads;
(2)SiO 2 @Fe 3 O 4 preparing magnetic beads:
taking 250mg of Fe 3 O 4 Placing the mixture into 20mL of absolute ethyl alcohol, performing ultrasonic treatment for 8.5min at the ultrasonic frequency of 35kHz, adding 28mL of ethyl alcohol, 58mL of ethanolamine and 5.26g of ultrapure water into a beaker, and uniformly stirring. Mixing the above liquid with Fe 3 O 4 Uniformly mixing the particles, putting the mixture into a three-neck flask, dropwise adding 0.5mL of tetraethyl orthosilicate (TEOS) while stirring at the rotating speed of 250rpm, adjusting the rotating speed to 450rpm after dropwise adding, mechanically stirring for 7h, washing the mixture with ethanol and deionized water for three times after the reaction is finished, and freeze-drying the mixture for 16h at the temperature of-78 ℃ to obtain SiO 2 @Fe 3 O 4 Magnetic beads, i.e., silica-coated magnetic beads;
(3)Ph-NH@SiO 2 @Fe 3 O 4 preparing magnetic beads:
taking 100mg of SiO 2 @Fe 3 O 4 Placing in 35mL anhydrous ethanol, ultrasonic dispersing for 13min with ultrasonic frequency of 35kHz, adding 90 μ L aniline methyl triethoxysilane (ND-42), mechanically stirring for 0.8h, washing with ethanol and deionized water for three times respectively after reaction, freeze drying for 22h at-78 deg.C to obtain Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads, i.e., magnetic bead complexes modified by anilination.
The magnetic bead composite modified by aniline prepared in this embodiment sequentially includes a ferroferric oxide core layer 1, a silica modification layer 2, and an aniline modification layer 3 from inside to outside.
Example 3
The preparation method of the magnetic bead complex modified by aniline concretely comprises the following steps:
(1)Fe 3 O 4 preparing magnetic beads:
0.746g (3.75 mmol) FeCl was weighed 2 ·4H 2 O and 2.16g (8.0 mmol) FeCl 3 ·6H 2 Dissolving O in 25mL deionized water, stirring at room temperature, setting the stirring speed at 500rpm/min, dropwise adding NaOH solution with the concentration of 32 w/v after all the solid is dissolved, generating black precipitate, continuously stirring for 1-2h to obtain black magnetic particles, washing the obtained black magnetic particles with deionized water until the pH is neutral, and freeze-drying at-72 ℃ for 26h to obtain black granular solid Fe 3 O 4 Magnetic beads;
(2)SiO 2 @Fe 3 O 4 preparing magnetic beads:
taking 250mg of Fe 3 O 4 Placing the mixture in 40mL of absolute ethyl alcohol for ultrasonic treatment for 7min, wherein the ultrasonic frequency is 45kHz, adding 32mL of ethyl alcohol, 62mL of ethanolamine and 5.3g of ultrapure water into a beaker, and uniformly stirring. Mixing the above liquid with Fe 3 O 4 Uniformly mixing the particles, putting the mixture into a three-neck flask, dropwise adding 0.7mL of tetraethyl orthosilicate (TEOS) while stirring at the rotating speed of 350rpm, adjusting the rotating speed to 550rpm after dropwise adding, mechanically stirring for 5 hours, washing the mixture with ethanol and deionized water for three times after the reaction is finished, and freeze-drying the mixture for 20 hours at the temperature of-74 ℃ to obtain SiO 2 @Fe 3 O 4 Magnetic beads, i.e., silica-coated magnetic beads;
(3)Ph-NH@SiO 2 @Fe 3 O 4 preparing magnetic beads:
taking 100mg of SiO 2 @Fe 3 O 4 Placing in 45mL anhydrous ethanol, ultrasonically dispersing for 17min at ultrasonic frequency of 45kHz, adding 110 μ L aniline methyl triethoxysilane (ND-42), mechanically stirring for 1.2h, washing with ethanol and deionized water for three times respectively after reaction, freeze drying for 26h at-74 deg.C to obtain Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads, i.e., magnetic bead complexes modified by anilination.
The magnetic bead composite modified by aniline prepared in this embodiment sequentially includes a ferroferric oxide core layer 1, a silica modification layer 2, and an anilino modification layer 3 from inside to outside.
Experimental example 1SEM image
Taking Fe prepared in example 1 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Three kinds of magnetic beads are respectively placed in absolute ethyl alcohol, uniformly dispersed by using ultrasonic waves to form turbid liquid, and the physical form, the dispersion state and the particle size parameters of the magnetic beads are observed by using a scanning electron microscope. Fe 3 O 4 Magnetic beads, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 SEM pictures of three kinds of magnetic beads are shown in FIG. 2, (a), (b) and (c) are Fe 3 O 4 Magnetic beads, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 As clearly shown in FIG. 2, the three kinds of magnetic particles are uniformly distributed, and have particle diameters of 10-20nm, 100-200nm and 100-200nm, respectively. Observing the morphology structure, and finding Fe in the structure 3 O 4 The surface of (a) is relatively rough; siO 2 2 @Fe 3 O 4 The surface of the magnetic bead is smoother, and the spherical shape is more obvious; ph-NH @ SiO 2 @Fe 3 O 4 The magnetic beads are more variable in shape. The changes in the particle size and morphology of the three types of magnetic beads fully account for the SiO 2 And anilino group has been successfully coated on Fe 3 O 4 The surface of the magnetic beads.
Test example 2X-ray diffraction Pattern
Taking Fe prepared in example 1 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Three magnetic beads are respectively and fully dried, then lightly ground into fine powder, and the components and the crystal forms of the magnetic particles are determined by adopting an X-ray diffraction (XRD) method. The recording range of the X-ray diffraction spectrum is 10.0000-80.0000 degrees, and the step size is 0.0200 degrees. The detection result of the X-ray diffraction is shown in FIG. 3, and the characteristic diffraction peak position of the sample is consistent with the standard parameter. For Fe 3 O 4 Characteristic peaks appear at 30.0 °, 35.5 °, 43.1 °, 57.1 °, 62.7 °; for SiO 2 @Fe 3 O 4 Characteristic peaks appear at 30.0 °, 35.3 °, 43.0 °, 57.2 °, 62.6 °; for Ph-NH @ SiO 2 @Fe 3 O 4 Characteristic peaks appear at 30.2 °, 35.5 °, 43.2 °, 57.1 °, 62.8 °, which correspond to the indices (220), (311), (400), (511) and (440) of the crystal planes in common at the diffraction angles, being Fe 3 O 4 Indicating that the magnetic particles produced are Fe 3 O 4 And the coating of the modifying group of the outer layer does not influence Fe 3 O 4 The crystal structure of (1).
Test example 3FT-IR spectrum
Taking Fe prepared in example 1 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Three magnetic beads, grinding the three magnetic beads into powder respectively, and measuring Infrared absorption signals of the three magnetic beads by a Fourier Transform Infrared Spectrometer (FTIR Spectrometer), wherein the wave number range is 4000-400 cm -1 The results are shown in FIG. 4. Wherein Fe 3 O 4 557cm in center -1 And SiO 2 @Fe 3 O 4 565cm in the middle -1 Is located at the Fe-O stretching vibration peak and SiO 2 @Fe 3 O 4 、Ph-NH@SiO 2 @Fe 3 O 4 Medium 457cm -1 、792cm -1 、1070cm -1 The positions are respectively a Si-O bending vibration peak, a Si-O-Si bending vibration peak and a Si-O stretching vibration peak, which indicate that SiO is 2 Has been successfully coated with Fe 3 O 4 The surface of the particles. Due to the preparation of Ph-NH @ SiO 2 @Fe 3 O 4 In the process of magnetic beads, few aminosilane coupling agents are added, so that anilino groups capable of being modified on the surfaces of the magnetic beads cannot be detected by using FT-IR. The surfaces of both beads were analyzed using an X-ray photoelectron spectrometer.
Test example 4X-ray photoelectron spectroscopy
Fe prepared in example 1 by X-ray photoelectron spectroscopy 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 The elementary analysis of the surfaces of three kinds of magnetic beads of the magnetic beads can be seen in FIG. 5, which shows that Ph-NH @ SiO 2 @Fe 3 O 4 Characteristic peaks of elements such as N and C exist in an XPS spectrum of the magnetic bead, wherein the content of the element of the N reaches 0.39%, and therefore it is proved that aniline groups are successfully modified on the surface of the magnetic bead in the preparation process.
Test example 5VSM curve
Testing of Fe prepared in example 1 3 O 4 Magnetic beads, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads the magnetic properties of the three magnetic beads were characterized at room temperature using a Vibrating Sample Magnetometer (VSM). As shown in FIG. 6, all three prepared magnetic beads have no hysteresis phenomenon, negligible residual magnetism and coercive force, good magnetic corresponding performance and strong superparamagnetism.
Test example 6Zeta potential Curve
Taking Fe prepared in example 1 3 O 4 Magnetic bead, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Three magnetic beads, namely three magnetic beads are placed in 50mL deionized water, the three magnetic beads are uniformly dispersed by ultrasonic treatment for 30min, and the Zeta potential of the magnetic particles is measured by a Malvern Nano ZS potentiometer, and the result is shown in figure 7. Finding Fe in solution 3 O 4 The Zeta potential of (b) is 19.4mV 2 @Fe 3 O 4 The Zeta potential is-19.2mV, ph-NH @ SiO 2 @Fe 3 O 4 Has a Zeta potential of 3.44mV, where SiO 2 @Fe 3 O 4 、Fe 3 O 4 The absolute value of the potential is obviously larger than Ph-NH @ SiO 2 @Fe 3 O 4 The absolute value of potential of (A), indicates Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads in aqueous solution to SiO 2 @Fe 3 O 4 、Fe 3 O 4 The magnetic beads are more prone to agglomeration.
Test example 7FFPE DNA extraction test
mu.L of lysis buffer pH 7 and 500. Mu.L of binding buffer pH 2 were added to each of the three tubes. 1500ng of FFPE DNA was added thereto, and 5. Mu.L of each of the three magnetic beads (0.5 mg) prepared in example 1 was added thereto, and the mixture was adsorbed at room temperature for 15min. And placing the three centrifuge tubes on a magnetic frame to clarify the solution, sucking the liquid into a new centrifuge tube, measuring the concentration by using the Qubit, and calculating the adsorption efficiency of the DNA. Then adding 600 mu L of 70% ethanol to wash the magnetic beads, uniformly mixing by vortex, then placing the magnetic beads on a magnetic rack again, after the solution is clarified, absorbing the liquid, and repeatedly washing once. After the flash separation, the liquid was blotted and the beads were dried at room temperature for 2min. Add 75. Mu.L of pH 8.0TE eluent, vortex, mix well and incubate at room temperature for 10min. And placing the centrifugal tube on the magnetic frame again, after the solution is clarified, sucking the liquid into a new EP tube, measuring the concentration by using the Qubit, and calculating the elution efficiency of the DNA.
As shown in FIG. 8, the extraction effect of FFPE DNA by the three magnetic beads is significantly different. Bare Fe 3 O 4 The magnetic beads have poor extraction effect on FFPE DNA, the adsorption rate reaches 73.6%, and the elution rate is only 12.6%. Through SiO 2 Modified SiO 2 @Fe 3 O 4 The extraction effect of the magnetic beads on the DNA is obviously improved, the adsorption efficiency is close to 100%, and the elution efficiency is 64.8%. Ph-NH @ SiO modified by anilino 2 @Fe 3 O 4 Although the adsorption capacity of the magnetic beads to DNA is reduced in the FFPE DNA extraction process, the elution efficiency is 72.1 percent, compared with SiO 2 @Fe 3 O 4 The magnetic beads increased by nearly 10%. Further, amino-modified NH prepared according to the same method 2 @SiO 2 @Fe 3 O 4 Although the capacity of adsorbing DNA is extremely strong and is close to 100%, the DNA hardly has elution capacity, and the difficulty of obtaining DNA from magnetic beads subsequently is extremely high. This shows that compared with the magnetic beads modified by amino groups, this shows that the magnetic beads have a certain improvement in the elution capability of DNA through further modification of the anilino groups.
The inventors also prepared Fe for other examples 3 O 4 Magnetic beads, siO 2 @Fe 3 O 4 Magnetic beads and Ph-NH @ SiO 2 @Fe 3 O 4 Magnetic beads the above experiments were carried out with three magnetic beads, which are substantially identical and not listed due to space limitations.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. The magnetic bead compound modified by phenylation is characterized by sequentially comprising a ferroferric oxide core layer, a silicon dioxide modification layer and an aniline group modification layer from inside to outside.
2. A preparation method of a magnetic bead compound modified by aniline is characterized by comprising the following steps:
(1) In FeCl 2 ·4H 2 O and FeCl 3 ·6H 2 Dripping a precipitator into the O mixed aqueous solution, washing and drying to obtain ferroferric oxide;
(2) Mixing the ferroferric oxide with ethanol, performing ultrasonic dispersion, adding a mixed solution of ethanol, ethanolamine and water, uniformly mixing, dropwise adding tetraethoxysilane under the condition of stirring, washing, and drying to obtain magnetic beads coated with silicon dioxide;
(3) Mixing the magnetic beads coated with the silicon dioxide with ethanol, performing ultrasonic dispersion, dropwise adding aniline methyl triethoxysilane under the stirring condition, washing, and drying to obtain the magnetic bead compound modified by aniline.
3. A method for preparing a complex of magnetic beads modified by anilination according to claim 2, wherein FeCl is used in step (1) 2 ·4H 2 The concentration of O in the mixed aqueous solution is 0.146-0.150mol/L, feCl 3 ·6H 2 The concentration of O in the mixed water solution is 0.28-0.32mol/L, and the precipitator is NaOH solution with the mass fraction of 28-32%.
4. The method for preparing a magnetic bead composite modified by anilination according to claim 2, wherein in step (2), the mass-to-volume ratio of ferroferric oxide to ethanol is 25g to 2L, the volume ratio of ethanol to ethanolamine to water in the mixed solution of ethanol to ethanolamine to water is 28 to 32.
5. The method of claim 2, wherein the ultrasonic dispersion time in step (2) is 7-10min, and the ultrasonic frequency is 35-45kHz.
6. The method for preparing a magnetic bead composite modified by phenylate according to claim 2, wherein in the step (2), the mass-to-volume ratio of the ferroferric oxide to the tetraethoxysilane is 250mg to 0.7mL, the stirring speed during the dropwise addition of the tetraethoxysilane is 250rpm to 350rpm, and after the dropwise addition is completed, the reaction is carried out at the stirring speed of 450rpm to 550rpm for 5 hours to 7 hours.
7. The method for preparing a magnetic bead composite modified by phenylation according to claim 2, wherein the mass-to-volume ratio of the silica-coated magnetic beads to ethanol in step (3) is 100mg, and the mass-to-volume ratio of the silica-coated magnetic beads to the aniline methyl triethoxysilane is 100mg.
8. A method for preparing a magnetic bead composite modified by anilino according to claim 2, wherein in the step (3), the aniline methyl triethoxysilane is added dropwise at a stirring speed of 450-600rpm, the stirring speed is kept unchanged, and the stirring reaction is continued for 0.8-1.2h to obtain the magnetic bead composite modified by anilino.
9. A method for preparing a magnetic bead composite modified by anilination according to claim 2, wherein in step (3), the ultrasonic dispersion time is 13-17min, and the ultrasonic frequency is 35-45kHz.
10. Use of the aniline modified magnetic bead complex according to claim 1 or the aniline modified magnetic bead complex prepared according to any one of claims 2 to 9 for extracellular free DNA extraction.
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Inventor after: Ye Feng Inventor after: Li Kun Inventor after: Xiao Lin Inventor after: Lv Xiaofang Inventor after: Cao Shiyu Inventor before: Ye Feng Inventor before: Li Kun Inventor before: Xiao Lin Inventor before: Cao Shiyu |