CN1284795C - Magnetic nanoparticle nucleic acid separator, its preparation method and application - Google Patents

Abstract

The present invention relates to a nucleic acid separator for magnetic nanometer particles, which comprises the following three layers of structures: (1) an inner nuclear layer which is composed of nuclear shell type magnetic nanometer particles; (2) a 3-triethoxysilyl trimethoxysilane intermediate layer coated on the inner nuclear layer; (3) a single-stranded DNA outer shell layer coated on the intermediate layer. The present invention also relates to a method for prparing the nucleic acid separator for magnetic nanometer particles. The nucleic acid separator for magnetic nanometer particles in the present invention can simply and effectively separate DNA or RNA of an object.

Description

Magnetic nanoparticle nucleic acid separator, its preparation method and application

Technical field

The invention relates to a nano biological separator, more specifically, the invention relates to a magnetic nano particle nucleic acid separator. The invention also relates to the preparation method and application of the separator.

Background technique

As we all know, the field of nanotechnology is the most popular field of science and technology research today. The emerging science and technology formed by applying nanotechnology to the field of biological sciences—nanobiotechnology is the use of nanotechnology to study and solve major problems in the field of life sciences. Science, it is becoming one of the current important frontier scientific research fields.

Nowadays, the application prospect of magnetic nanoparticles in the field of biotechnology has attracted increasing attention. Magnetic nanoparticles are often used as separation media for magnetic field separation of biological samples. The separation method using magnetic nanoparticles is easy to operate, the equipment required is cheap, and the separation speed is fast at the same time, which is conducive to maintaining the biological activity of the sample. At present, it is more and more widely used.

Magnetic nanoparticles used in biotechnology are generally required to have the following characteristics: (1) the particles are superparamagnetic and easy to adsorb and elute; (2) the particles require a large magnetization to ensure the sensitivity of the separation efficiency; ( 3) The particle surface should be easy to be chemically modified to connect with different biological and drug molecules; (4) It requires better biocompatibility when used in vivo. Nanoparticle separators designed for this purpose usually have a sandwich structure. The inner core layer is generally made of magnetic nanomaterials, and the middle is a coating layer. Most of the existing products currently use polymer materials as the coating layer, and the outermost layer is modified with Biological tissues have functional groups with specific effects to meet the requirements of biological separation.

However, the separators in the prior art mostly use polymer microspheres obtained by emulsion polymerization, which are coated with nanometer-sized magnetic particles. However, the structure of polymer microspheres is usually relatively loose, and the magnetic particles contained in it are easy to break away from the microspheres and agglomerate during long-term storage, thereby affecting the performance of the product; using adsorption or affinity on magnetic nanoparticles When fixing the active substance, it is easy to fall off.

The invention provides a magnetic nanoparticle nucleic acid separator, which overcomes the shortcomings of the prior art.

Contents of the invention

The object of the present invention is to provide a magnetic nano particle nucleic acid separator.

Another object of the present invention is to provide a method for preparing the magnetic nanoparticle nucleic acid separator.

Another object of the present invention is to apply the magnetic nanoparticle nucleic acid separator to the separation of target DNA or RNA.

In a first aspect of the present invention, a magnetic nanoparticle nucleic acid separator is provided, which contains the following three-layer structure:

(1) an inner core layer composed of core-shell magnetic nanoparticles;

(2) MPTS (3-mercaptopropyltrimethoxysilane) intermediate layer covering the core layer;

(3) Single-stranded DNA outer layer covering the middle layer.

In another preferred example, the core layer of the core-shell magnetic nanoparticle includes an inner core and an outer shell. The inner core composition of the inner core layer is selected from: iron oxide, nickel, nickel-iron alloy, or a combination thereof. Preferably, the core component of the core layer is iron oxide. The shell composition of the inner core layer is selected from silica, agarose, olefin polymer, polyacrylonitrile, epoxy compound, or a combination thereof. Preferably, the outer shell component of the inner core layer is silicon dioxide.

Wherein, the single-stranded DNA outer layer is a single-stranded DNA connected to the middle layer through a persulfide bond.

In a second aspect of the present invention, a method for preparing a magnetic nanoparticle nucleic acid separator is provided, comprising the following steps:

(1) In a microemulsion system containing magnetic nanoparticles and an inner shell shell forming agent (such as tetraethyl orthosilicate), a core-shell magnetic nanoparticle is formed by a water-in-oil type inverse microemulsion method, the described Core-shell magnetic nanoparticles include a core and a shell,

The magnetic nanoparticles are selected from: iron oxide, nickel, nickel-iron alloy, or a combination thereof;

The inner-shell-forming agent-forming ingredients are shells selected from the following group: silicon dioxide, agarose, olefin polymers, polyacrylonitrile, epoxy compounds, or combinations thereof;

(2) modifying the surface of the core-shell magnetic nanoparticles in step (1) with 3-mercaptopropyltrimethoxysilane to obtain magnetic nanoparticles modified with mercapto groups;

(3) Using single-stranded DNA modified with persulfide bonds to modify the surface of the magnetic nanoparticles modified with sulfhydryl groups in step (2) to form a magnetic nanoparticle nucleic acid separator.

Wherein, TritonX-100, n-hexanol and cyclohexane in the microemulsion system are evenly mixed in a ratio of 1:(1-3):(4-6).

In the third aspect of the present invention, the use of the magnetic nanoparticle nucleic acid separator of the present invention is provided, which is used for separating DNA or RNA.

In a fourth aspect of the present invention, a method for isolating nucleic acid is provided, comprising the steps of: adding the magnetic nanoparticle nucleic acid separator to a solution containing target single-stranded DNA or RNA, making it dissolve in Tris-HCl and MgCl ₂ After fully hybridizing at a certain temperature in a mixed solution, the target single-stranded DNA or RNA is separated under the guidance of an external magnetic field.

Description of drawings

Figure 1 is a surface-enhanced Raman effect diagram of a magnetic nanoparticle (FSM) modified with a thiol group. Raman enhancement effect.

Figure 2 is a surface-enhanced Raman effect diagram of magnetic nanoparticles (FSMD) connected to single-stranded DNA, which shows that DNA is connected to the surface of magnetic nanoparticles and has a better Raman enhancement effect.

Detailed ways

After extensive and in-depth research, the inventors have invented substances that can recognize and bind DNA or RNA on the surface of magnetic nanoparticles, and then use this magnetic nanoparticle to capture target DNA or RNA, and then carry out the process under the action of an external magnetic field. separate technology.

The method for preparing magnetic nanoparticle nucleic acid separator of the present invention comprises the following steps:

(1) Preparation of magnetic nanoparticles

The mixed solution of FeSO ₄ ·7H ₂ O and FeCl ₃ ·6H ₂ O and the NaOH solution were prepared respectively with twice distilled water. The concentration of Fe ²⁺ ions in the iron salt mixed solution is 0.1-0.2 mol/l, the concentration of Fe ³⁺ ions is 0.1-0.3 mol/l, and the concentration of NaOH solution is 2-3 mol/l. Under vigorous stirring, the NaOH solution whose volume is half of the volume of the mixed salt solution was slowly added dropwise into the mixed salt solution. Aging the obtained solid precipitate at 40°C to 60°C for 12 hours, washing the precipitate several times with double distilled water, filtering and drying at 40°C to 80°C for 24 hours, grinding it in an agate mortar That is the product.

(2) Modification of silica on the surface of γ-Fe ₂ O ₃

Mix TritonX-100, n-hexanol and cyclohexane uniformly in a ratio of 1:(1-3):(4-6) to form a transparent and stable microemulsion system. Put the above-mentioned microemulsion system in ultrasonic treatment for 30-60 minutes, then add 0.01-1g of γ-Fe ₂ O ₃ (magnetic nanoparticles) to it, treat it with ultrasonic wave for 3 minutes, take out the supernatant liquid and pour it into a three-necked flask , Stir for 30-60 minutes to make it even. Take 1ml of a certain concentration of concentrated ammonia water and dilute it with 2ml of double distilled water, slowly add it into the microemulsion that is constantly stirring, and keep stirring for 10 to 60 minutes to make the ammonia water evenly dispersed in the microemulsion. After 1 hour, add 1-5 ml of orthosilicate ethyl ester (inner shell shell forming agent) dropwise to the microemulsion, while stirring continuously for 10 hours, and keep the temperature of the system between 15-40°C. Add acetone to the system to precipitate the particles, or let the system stand overnight to allow the particles to settle naturally, and use ethanol to wash the particles. Calcining the cleaned particles at 300-700° C. for 1-4 hours to collect core-shell magnetic nanoparticles.

(3) Modification of the surface of magnetic nanoparticles with 3-mercaptopropyltrimethoxysilane (MPTS)

Take 15mg of magnetic nanoparticles and add them to the mixture of acetic acid and ethanol in 2-10ml, and treat them with ultrasonic waves for 20-60 minutes; weigh 0.01-1g MPTS, and treat them with ultrasonic waves for 10-60 minutes; React at 10-40°C for 1 hour, then take out the particles and wash them three times with a mixture of acetic acid and ethanol, then vacuum-dry them at 100-300°C for 2 hours to collect the particles (FSM).

(4) Modification of single-stranded DNA on the surface of magnetic nanoparticles

Take a certain amount of single-stranded DNA that has been modified with persulfide bonds, add it to 500 μl of NaHCO ₃ and Na ₂ CO ₃ mixture, mix well, add a small amount of FSM to the solution of single-stranded DNA to disperse it Uniform, and react in a humid environment at 10-50°C for 12-36 hours (the persulfide bond on the DNA reacts with the sulfhydryl group on the middle layer, so that the DNA molecule is directly connected to the middle layer through the persulfide bond). The cleaning particles are separated and vacuum-dried under the condition of 20-80°C for 10 hours, and then the particles are dispersed (FSMD) with double distilled water to obtain a magnetic nano-particle nucleic acid separator.

The length of single-stranded DNA suitable for use in the present invention is not particularly limited. Usually the average length is about 5-200 bp, preferably about 10-50 bp.

The core component of the core layer of the core-shell magnetic nanoparticle includes nickel (Ni) or an alloy of nickel (Ni) and iron (Fe) in addition to iron oxide.

In addition to inorganic coating layers such as silicon dioxide, the shell components of the core layer of core-shell magnetic nanoparticles also include organic coating layers such as agarose, olefin polymers, polyacrylonitrile, and epoxy compounds. For selected shell components, suitable core layer shell forming agents can be selected according to the prior art. For example, when the shell component is silica, tetraethyl orthosilicate or other suitable core shell formers may be used.

The main advantages of the present invention are:

(1) The magnetic nanoparticles used are monodisperse, do not produce agglomeration, and the shape and diameter of the magnetic nanoparticles are easy to control;

(2) The single-stranded DNA that can recognize the target DNA or RNA is connected to the magnetic nanoparticles through chemical bonds, which has good firmness and is not easy to fall off;

(3) Compared with the traditional electrophoresis method, the method of the present invention is simple and efficient.

Below in conjunction with specific embodiment, the present invention will be further elaborated. It should be understood that the present invention is not limited to these specific examples.

Example 1

Preparation method of inner core layer of magnetic nanoparticles

The core layer of the magnetic particle is prepared by improved chemical co-precipitation, and the specific method is as follows: a mixed solution of FeSO ₄ ·7H ₂ O and FeCl ₃ ·6H ₂ O and a NaOH solution are respectively prepared with double distilled water. The concentration of Fe ²⁺ ions in the iron salt mixed solution is 0.1-0.2 mol/l, the concentration of Fe ³⁺ ions is 0.1-0.3 mol/l, and the concentration of NaOH solution is 2-3 mol/l. Under vigorous stirring, the NaOH solution whose volume is half of the volume of the mixed salt solution was slowly added dropwise into the mixed salt solution. Aging the obtained solid precipitate at 40°C to 60°C for 12 hours, washing the precipitate several times with double distilled water, filtering and drying at 40°C to 80°C for 24 hours, and grinding it in an agate mortar Get the product.

Example 2

Preparation method of core-shell nucleic acid magnetic nanoparticles

Mix TritonX-100, n-hexanol and cyclohexane uniformly at a ratio of 1:2:5 to form a transparent and stable microemulsion system. Put the above microemulsion system in the ultrasonic treatment for 30-60 minutes, then add 0.5g of γ-Fe ₂ O ₃ to it, after 6 minutes of ultrasonic treatment, take out the supernatant liquid and pour it into a three-necked flask, stir for 30 minutes to make it uniform. Take 1ml of a certain concentration of concentrated ammonia water and dilute it with 2ml of double distilled water, slowly add it into the microemulsion that is constantly stirring after 30 minutes, and keep stirring for 30 minutes to make the ammonia water evenly dispersed in the microemulsion. After 1 hour, 1-3 ml of tetraethyl orthosilicate was added dropwise to the microemulsion, while stirring continuously for 10 hours, and the temperature of the system was kept between 15-30°C. Add acetone to the system to precipitate the particles, or let the system stand overnight to allow the particles to settle naturally, and use ethanol to wash the particles. The cleaned particles are placed under the condition of 400-700° C., calcined for 1-4 hours, and the particles are collected.

Example 3

Preparation of Magnetic Nanoparticle Nucleic Acid Separator

Get 15mg of the magnetic nanoparticles prepared in Example 2 and add 2ml of the mixed solution of acetic acid and ethanol, and use ultrasonic treatment for 20 to 60 minutes; weigh 0.01g MPTS, and use ultrasonic treatment for 10 to 60 minutes; Mix evenly, react at 10-30°C for 1 hour, then take out the particles and wash them three times with a mixture of acetic acid and ethanol, then vacuum-dry them at 100-300°C for 2 hours to collect the particles (FSM). It can be seen from Figure 1 that the sulfhydryl groups are modified on the surface of magnetic nanoparticles and have a good Raman enhancement effect.

Take a certain amount of single-stranded DNA (average length 7bp) that has been modified with a persulfide bond, add it to 500 μl of NaHCO ₃ and Na ₂ CO ₃ mixture, mix well, and add a small amount to the single-stranded DNA solution FSM, make it dispersed evenly, and react for 24 hours under the condition of 20-40°C in a humid environment. The cleaning particles are separated and vacuum-dried under the condition of 20-80°C for 10 hours, and then the particles are dispersed (FSMD) with double distilled water to obtain a magnetic nano-particle nucleic acid separator. It can be seen from Figure 2 that the DNA probes are connected to the surface of magnetic nanoparticles, and there is an obvious Raman enhancement effect.

Example 4

Preparation of Magnetic Nanoparticle Nucleic Acid Separator

Take 15 mg of the magnetic nanoparticles prepared in Example 2 and add 6 ml of the mixed solution of acetic acid and ethanol, and use ultrasonic treatment for 20-60 minutes; weigh 0.5 g MPTS, and use ultrasonic treatment for 10-60 minutes; mix the two solutions Evenly, react at 10-30°C for 1 hour, then take out the particles and wash them three times with a mixture of acetic acid and ethanol, then vacuum-dry them at 100-300°C for 2 hours, and collect the particles (FSM).

Take a certain amount of single-stranded DNA (average length 50bp) that has been modified with a persulfide bond, add it to 500 μl of NaHCO ₃ and Na ₂ CO ₃ mixture, mix well, and add a small amount to the solution of single-stranded DNA FSM, make it dispersed evenly, and react for 24 hours under the condition of 20-40°C in a humid environment. The cleaning particles are separated and vacuum-dried under the condition of 20-80°C for 10 hours, and then the particles are dispersed (FSMD) with double distilled water to obtain a magnetic nano-particle nucleic acid separator.

Example 5

Preparation of Magnetic Nanoparticle Nucleic Acid Separator

Take 15 mg of the magnetic nanoparticles prepared in Example 2 and add them to the mixed solution of 10 ml of acetic acid and ethanol, and use ultrasonic treatment for 20 to 60 minutes; weigh 1 g of MPTS, and use ultrasonic treatment for 10 to 60 minutes; mix the two solutions evenly , react at 10-30°C for 1 hour, then take out the particles and wash them three times with a mixture of acetic acid and ethanol, then vacuum-dry them at 100-300°C for 2 hours to collect the particles (FSM).

Take a certain amount of single-stranded DNA (average length 95bp) that has been modified with persulfide bonds, add it to 500 μl of NaHCO ₃ and Na ₂ CO ₃ mixture, mix well, and add a small amount to the solution of single-stranded DNA FSM, make it dispersed evenly, and react for 24 hours under the condition of 20-40°C in a humid environment. The cleaning particles are separated and vacuum-dried under the condition of 20-80°C for 10 hours, and then the particles are dispersed (FSMD) with double distilled water to obtain a magnetic nano-particle nucleic acid separator.

Example 6

Take the magnetic nanoparticle nucleic acid separator prepared in Examples 3-5, add 100 μl of total RNA aqueous solution, and then add 20-50 μl of sodium citrate buffer, mix well, and hybridize at 10-50° C.

Collect the magnetic nanoparticles under the action of an external magnetic field and remove the supernatant. Wash the magnetic nanoparticles three times with sodium citrate buffer. Suspend the magnetic nanoparticles in water, remove the magnetic nanoparticles under the action of an external magnetic field, collect the aqueous phase, and store it at -80°C for future use or for RT-PCR, cDNA library construction, etc.

Results: The core-shell nucleic acid magnetic nanoparticles prepared in Examples 3-5 are monodisperse, no agglomeration phenomenon occurs, and their shape and diameter are easy to control. In addition, the single-stranded DNA that can recognize the target DNA or RNA is connected to the magnetic nanoparticles through chemical bonds, which has good firmness and is not easy to fall off, so the separation effect is very good.

Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (7)

1. magnetic nano-particle separate nucleic acid device is characterized in that it contains following three-decker:

(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle, wherein, described core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell, the interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination; The outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;

(2) the 3-sulfydryl propyl trimethoxy silicane middle layer of coating inner nuclear layer;

(3) the single stranded DNA outer shell in coating middle layer.

2. magnetic nano-particle separate nucleic acid device according to claim 1 is characterized in that the interior nuclear composition of described inner nuclear layer is the oxide compound of iron.

3. magnetic nano-particle separate nucleic acid device according to claim 1 is characterized in that the outer shell component of described inner nuclear layer is a silicon-dioxide.

4. magnetic nano-particle separate nucleic acid device according to claim 1 is characterized in that, described single stranded DNA outer shell is by crossing the single stranded DNA that sulfide linkage is connected with the middle layer.

5. method for preparing magnetic nano-particle separate nucleic acid device is characterized in that it may further comprise the steps:

(1) in the microemulsion system that contains magnetic nano-particle and inner nuclear layer shell formation agent, by the water-in-oil-type reverse microemulsion process, form the core-shell type magnetic nano particle, described core-shell type magnetic nano particle comprises kernel and shell,

Described magnetic nano-particle is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination;

Described inner nuclear layer shell forms agent and is formed into the shell that branch is selected from down group: silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;

(2) use 3-sulfydryl propyl trimethoxy silicane that the core-shell type magnetic nano particle surface of step (1) is modified, obtained modifying the magnetic nano-particle of sulfydryl;

(3) use modified the single stranded DNA of crossing sulfide linkage to the modification of step (2) surface of magnetic nano-particle of sulfydryl modify, form magnetic nano-particle separate nucleic acid device.

6. method according to claim 5 is characterized in that, TritonX-100 in the described microemulsion system, n-hexyl alcohol, hexanaphthene are in 1: 1～3: 4～6 ratio uniform mixing.

7. the purposes of magnetic nano-particle separate nucleic acid device as claimed in claim 1 is characterized in that, is used for DNA isolation or RNA.

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