JPH03242327A - Production of magnetic fine particle - Google Patents

Abstract

PURPOSE:To obtain magnetic fine particles having an uniform particle diameter and particle size distribution capable of uniformly dispersing into a solution by making total value of Fe ion concentration in an aqueous solution of raw material containing Fe<2+> and/or Fe<3+> and an organic coating layer-forming material to below a specific value and increasing pH with heat decomposition of added urea. CONSTITUTION:Total value of Fe ion concentration in an aqueous solution of raw material containing Fe<2+> and/or Fe<3+> and an organic coating layer- forming material (e.g. polysaccharide such as dextran) is made to below 6X10<-2>mol/l. Next, urea is added to the aqueous solution of raw material and said urea is decomposed by heat, then pH of the aqueous solution is uniformly increased, e.g. to pH8 by generated NH3. By said method, the aimed magnetic fine particles of magnetite having <=150Angstrom particle diameter and organic coating layer on the surface is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing magnetite magnetic fine particles on which an organic coating layer for labeling biological materials is formed.

[Prior Art and its Problems] Magnetic particles have been conventionally used as magnetic fluids and magnetic inks because they can be selectively separated, collected, and moved using a magnetic field after being bound to a specific object. Recently, it has been proposed to use it as a carrier for biopolymers, cells, viruses, or drugs.

Magnetic particles intended for binding to minute objects such as cells and viruses are desired to have improved properties compared to those conventionally used in magnetic fluids.

For example, in the case of magnetic particles designed to bind to viruses, it is desirable that the particle size is controlled to about 100 particles, and that the particle size distribution is close to monodisperse.

This particle size is a value when magnetite (PE304) is considered, but this is a value equivalent to the size of a virus (it easily binds to a virus), and is also a value that satisfies the magnetic properties. In addition, each magnetic fine particle must have an appropriate organic coating layer formed on its surface so that it is difficult to aggregate, and must be uniformly dispersed in the solution.

As a method for producing the above-mentioned magnetic fine particles that capture microscopic biomaterials, attempts have been made to synthesize iron oxide in an aqueous solution of dextran, which is a polysaccharide, or in liposomes, which are lipids.

In a previous patent application, the present inventor also found that by adding aqueous ammonia to a dextran aqueous solution such that the molar ratio of divalent and trivalent iron ions was 2 Fe"<Fe", the average particle size was 100. It has been reported that it is possible to synthesize Fe3O4 microparticles with a size of , or larger.

It has been clarified that the magnetic fine particles obtained in this manner have zero residual magnetization and exhibit superparamagnetism because their surfaces are coated with dextran.

Although the above manufacturing method was satisfactory in controlling the magnetic properties and average particle size of the obtained magnetic fine particles, the particle size distribution and uniform dispersibility of the magnetic fine particles in a solution were not necessarily satisfactory. It wasn't.

Conventionally, the magnetic fine particles used for biomaterials are R, S, Mo1da.
and Mackenzie, J. Immunol.
logical Methods 52p353.198
It was manufactured by the method described in 2.

This method involves dropping ammonia water into an aqueous solution containing dextran, FeCQ3, and FeC(h).
The pH of the aqueous solution is increased to generate magnetite fine particles in the solution. However, this method has the disadvantage that the pH of the area where the aqueous ammonia is dropped suddenly changes, and only a fine particle dispersion with poor uniformity can be obtained.

On the other hand, in order to achieve a homogeneous reaction, attempts have been made to synthesize magnetite for use in recording media by adding urea to an aqueous iron chloride solution in advance, and increasing the temperature to decompose it and generate ammonia. However, when this method using urea is applied to the synthesis method in the above literature, 30
There was a problem in that magnetite fine particles of 0 to 400 A precipitated all at once, making it impossible to obtain dextran-coated fine particles. This is because decomposition of urea requires a temperature of 80 to 90° C., which is close to the boiling point of water, and therefore the particle size of the generated fine particles cannot be kept small. The magnetite obtained in this way no longer exhibits superparamagnetism, and therefore cannot be used as magnetic fine particles for labeling.

An object of the present invention is to provide magnetic microparticles that have a uniform particle size and particle size distribution and can be uniformly dispersed in a solution and are suitable for use in microbiological material labels.

[Means for Solving the Problems] The production method of the present invention increases p) of a raw material aqueous solution containing divalent iron ions and/or trivalent iron ions and an organic coating layer forming material to form an organic coating on the surface. A method for manufacturing magnetic fine particles in which a layer is formed, in which the total iron ion concentration of the raw material aqueous solution is 6 X I O-'moI: l/
(L or less and adding urea to the raw material aqueous solution,
The solution was to produce magnetite magnetic @ particles with a particle size of 150 Å or less by thermally decomposing this urea and increasing the pH of the raw material aqueous solution.

[Function] In the production method of the present invention, fine magnetite particles are synthesized by uniformly increasing the pH of the raw material aqueous solution using ammonia generated by thermal decomposition of urea.

In addition, by limiting the ratio of the total concentration of divalent and trivalent iron ions in the raw material aqueous solution to the concentration of the organic coating layer forming material, it is possible to prevent the obtained magnetic fine particles from increasing in particle size too much and settling. .

Examples of the organic coating layer forming material used in the production method I of the present invention include polysaccharides such as dextran, lipids such as liposomes, and water-soluble polymers such as polyvinyl vinyl alcohol.

Note that the solubility of the organic coating layer forming material in water is generally not large, and is, for example, about 25 g dextran/100 g HxO in the case of dextran (D).

Therefore, in the present invention, a practical method was adopted in which the concentration of the organic coating layer-forming material was selected to be a constant value close to the solubility limit at which it could be dissolved in water, and the concentration of iron ions was set in the optimum concentration range.

Further, in the present invention, the iron ions contained in the raw material aqueous solution are p
There are no particular limitations as long as magnetite fine particles are precipitated by an increase in H, and divalent iron ions (Fe 2 + ) and trivalent iron ions (p e ! +
) may be contained.

[Example code] Hereinafter, the present invention will be described in detail with reference to Examples.

(Example 1) The heating time of the raw material aqueous solution is plotted on the horizontal axis, and the pH change of the aqueous solution is plotted on the vertical axis, and the results are shown in FIG.

Note that the manufacturing conditions here are as follows.

Iron ion concentration: 4.4 x 10-3 mol: l/(
IF e”:F e”= I :2 Urea concentration・IOg/i! Dextran concentration: 10g/ρ Pour this raw material aqueous solution into a sealed Teflon container, approx.
They were placed in an oil bath at 10°C, removed after a certain period of time, and analyzed one after another.

Here, the dextran concentration was set low to demonstrate the generation, growth, and sedimentation of magnetite fine particles that occur in the conventional method. The manufacturing conditions in the Mo1day document mentioned above are iron ion concentration: 2.2 x 10
-'mot2/(1, dextran concentration: 125g/C
It is.

Even if a similar experiment is carried out by adding urea to an aqueous solution of this concentration, a phenomenon similar to the result shown in FIG. 1 is observed.

Approximately 50 minutes after heating, the yellow color of the iron chloride aqueous solution changes to black. At this time, the solution was taken out, dried, and analyzed by X-ray diffraction, and it was found that magnetite with a particle size of about 70 mm was produced. Heating was continued further, and after about one hour, fine particles began to settle. These fine particles continued to grow even after settling, and the particle size increased to 175 particles after one and a half hours and to 228 particles after two hours.

Under these heating conditions, after about 2 hours, the value of p) becomes almost saturated.

The iron ion concentration in Example 1 (4,4X l O-3mo(
It has become clear that in order to prevent precipitation at 1/(1), the concentration of dextran must be at least 50 g/(!).

In Example I, the urea concentration is IOg/(!, but this is because the concentration of iron chloride is relatively low. When producing magnetic fine particles by the production method of the present invention, the iron chloride aqueous solution is acidic. Therefore, it is necessary to add urea appropriately depending on the iron ion concentration.

The results are shown in FIG. 2, with the horizontal axis representing the molar ratio of urea and iron ions, and the vertical axis representing the pH of the iron chloride aqueous solution after heating. When the molar ratio of urea and iron ions exceeds 50, pH = 8.
This indicates that the pH will become saturated from now on. Therefore, it can be seen that the molar ratio of urea to iron ions in the raw material aqueous solution needs to be at least 50 or more. Note that Example I
The molar ratio of urea to iron ions is 189, which satisfies the conditions shown in FIG.

(Example 2) An aqueous solution sample containing dextran-coated magnetite fine particles was prepared by the production method of the present invention and the conventional production method (the method described in the Molday literature). Then, it was measured how uniformly the magnetite particles were dispersed in each aqueous solution sample without agglomeration.

The manufacturing and measurement steps will be explained in detail below.

First, an aqueous solution sample was prepared using the production method of the present invention.

Iron ion concentration (Fe":Fe"-1:2): 2.2
Xl 0-2mo (1/L 1 , I
O-2mob/ 12.7.3×10-3mo(1/Q
, 4.4 x 10-”mo(1/(1,2,2×
10-3 moI2/ (100 g each in 1 aqueous solution
/12 dextran (weight average molecular weight: 40,000)
and 50 g/ρ of urea were added to prepare a raw material aqueous solution. Pour each raw material aqueous solution into a Teflon container that can be sealed, 110. 'C oil bath for 2 hours.

Thereafter, each raw material aqueous solution was dialyzed and diluted with pure water so that the iron ion concentration was 2.2 x 1.0-3 mo (1/Q).

Next, an aqueous solution sample was prepared using a conventional method.

Iron ion concentration (Fe”:Fe”=1:2): 4.4
X10-'mo&/12 aqueous solution IC10m&, 2
5 g of dextran (weight average molecular weight: 1:40000) was added to prepare a raw material aqueous solution. 7.5% ammonia water was added dropwise to this raw material aqueous solution. During the dropwise addition, sufficient stirring was performed while maintaining the solution temperature at 5°C. Thereafter, it was aged at 60°C for 1 hour. This solution was dialyzed, and at the time of measurement, the iron ion concentration was 2.2
o(Diluted with pure water to 1/Q.

Comparing the aqueous solution sample obtained by the present invention and the aqueous solution sample obtained by the conventional method, r=.

Regarding organic matter-coated ultrafine particles, it is not always clear how to measure the dispersibility in the following manner. -The method known for boat fishing is transmission electron microscopy (TEM).
However, it is impossible to directly measure an aqueous solution. For this reason, a method has been adopted in which some of the particles in the aqueous solution are scooped onto a mesh, and the state of the particles in the aqueous solution is inferred from the state of agglomeration of the particles on the mesh. Since this method is indirect and difficult to express numerically, we will discuss here the angular dependence of light scattering, the results of dynamic light scattering, and TEM.
By comparing the results with the observation results, we were able to more directly understand the dispersion state of the particles.

Figure 3 shows a schematic diagram of the light scattering measurement system. This measurement method uses a cell filled with an aqueous solution containing fine particles with a wavelength of 0.4
It irradiates with 48 μm light (argon laser) and measures the amount of scattered light while changing the angle. This scattered light has the same frequency as the incident light, and this phenomenon is called static light scattering. Then, the static light scattering of a Hensen's solution or the like is measured in advance as a standard, and the amount of scattering of the sample is determined by comparing it with that value.

Specifically, R(θ)-(r"Iθ)/(V Io)r: distance from scattering center to observation surface, ■θ・scattered light intensity, I., incident light intensity, θ: scattering angle ,
■= It is determined as the Rayleigh ratio expressed by the scattering volume.

FIG. 4 shows the Rayleigh ratio with respect to the scattering angle of each aqueous solution sample. The numerical values in the figure indicate the iron ion concentration of the raw material aqueous solution of each aqueous solution sample, and the concentration of the aqueous solution sample is converted to iron ions by 2, 2 x 10-
"moQ/(l). From Figure 4, it can be seen that as the iron ion concentration of the raw material aqueous solution decreases, the amount of scattering decreases and forward scattering also decreases. The diagram becomes complicated. Therefore, all aqueous solution samples are not shown in the figure, but the iron ion concentration of the raw material aqueous solution is 2.
10-'mo(1/(1 (1/10 concentration of conventional example)
In the sample aqueous solution, sedimentation was observed in some of the particles, and the scattering values were approximately the same or slightly higher than the results of the conventional method.

In addition, the iron ion concentration of the raw material aqueous solution ranged from 1.1XIO-2 (1/20 concentration of the conventional example) to 2.2 x 10-3mo.
(Up to 1/ρ (concentration 1/100 of the conventional example), a certain proportional relationship was observed between the iron ion concentration and the amount of scattering.

After placing the fine particles in these aqueous solution samples on a mesh for a certain period of time, the moisture was absorbed with a filter paper and observed with a TEM (approximately 20,000 times magnification). It became clear that there was a correlation. This also revealed that the dispersibility of organic substance-coated fine particles in a solution can be evaluated by static light scattering.

For comparison, FIG. 4 also shows the scattering characteristics of an aqueous suspension of all fine particles (commercially available), which is well known to be monodispersed. The Rayleigh ratio of this total fine particle suspension aqueous solution is 2.2 × 10 of the aqueous solution sample according to this example.
It is closest to the example of −3 mol/(l, and is characterized by almost no forward scattering.

From this, by applying the manufacturing method of the present invention,
It has become clear that magnetite fine particles can be obtained in a state of dispersion comparable to that of gold y1 particles.

Furthermore, a dynamic light scattering method is known for more directly measuring the particle size distribution of fine particles. This dynamic light scattering method uses Doppler shift to observe whether light with a frequency different from that of the incident light is detected in the light scattered when a laser tip is irradiated with fine particles in a solution undergoing Brownian motion. The wavelength of the incident light is about 1/1 of the wavelength of the incident light.
It is possible to evaluate 100 particle sizes (nm order).

Next, the sample aqueous solution was ultrafiltered to sufficiently remove excess dextran, and then the particle size distribution of the dextran-coated magnetite fine particles was measured by a dynamic scattering method. The measurement results are shown in FIG.

From FIG. 5, as the initial concentration of iron ions in the raw material aqueous solution increases, the particle size of the obtained fine particles also increases.
It was found that the distribution was wide. It is thought that this does not represent the particle size of a single fine particle, but rather the apparent particle size when several fine particles aggregate. This result is consistent with the tendency of static light scattering mentioned earlier and the T
This supports the EM observations.

(Example 3) In order to clarify the influence of the concentration of dextran added to the raw material aqueous solution on the dispersibility of fine particles, an experiment was conducted under the following conditions.

Iron ion concentration of raw material aqueous solution・2,2XIO-3
mo(!/12F e”:F e”= 1 : 2 Amount of urea added: 25 g/l! Keeping the other conditions of the raw material aqueous solution constant, the dextran concentration is 25 g/L, 50 g/&, 100 g/
Figure 6 shows the scattering properties of the aqueous solution samples obtained by changing the dextran concentration. From Figure 6, it was confirmed that as the dextran concentration increased, the light scattering decreased and the dispersion properties improved. By setting the dextran concentration to about 100 g/Q or more, the Rayleigh ratio is 5 x 10-5c.
It was found that a magnetite fine particle suspension solution having excellent dispersibility (comparable to gold fine particles) of less than m-' can be obtained.

Moreover, FIG. 7 shows the change in the amount of light scattering when the amount of urea added to the raw material aqueous solution was changed. The conditions for producing the raw material aqueous solution are as follows.

Iron ion concentration of raw material aqueous solution: 2.2 x 10-”m
oQ/(IF e": Fe"=1:2 Dextran concentration: 100 g/l From FIG. 7, it was found that the higher the urea concentration, the better the dispersibility and the less light scattering could be obtained.

(Example 4) In order to clarify what particle size of magnetite is synthesized by the production method of the present invention, an experiment was conducted using the following parameters.

Dextran concentration (molecular weight 40000) 125 g/
(l (value given in Molday's literature), 250g/&
Urea concentration: 50g/C Iron ion concentration of raw material aqueous solution: IO-3 to IO-'
moQ#F e":F e"-1: 2 The particle size of the produced fine particles was determined by measuring the diffraction peak due to magnetite by X-ray diffraction, and determining it from the half width (SCherrer's formula).

However, if the initial concentration of iron ions is on the order of lo-3, the amount of magnetite produced is too small compared to dextran, making measurement by X-rays difficult. Therefore, the prepared solution must be concentrated by ultrafiltration. After that, it was dried at 40°C to prepare a measurement sample.

This ultrafiltration step removes dextran that is not bound to magnetite (referred to as free dextran). FIG. 8 shows the X-ray diffraction patterns of the samples when the initial concentration of iron ions was changed. The large halo pattern is derived from dextran, and the magnetite peak appears overlapping it. The particle size was evaluated using (311), which is the largest diffraction peak. The upper two diffraction patterns in FIG. 8 are for a sample that is uniformly dispersed in the solution, and the lower diffraction pattern is for a sample that has precipitated. It can be seen that in the precipitated magnetite, the organic coating layer of dextran is extremely thin.

Further, the horizontal axis represents the initial concentration of iron ions, and the vertical axis represents the particle size of the produced magnetite fine particles, and the relationship is shown in FIG. 9. In Figure 9, the white circle indicates the dextran concentration of 125.
In the case of g/(, the black circles represent the case of 250 g/ρ. As the iron ion concentration of the raw material aqueous solution increases, the particle size increases, and when the particle size reaches a certain value, sedimentation begins. Then, the concentration of dextran increases. As a result, the particle size tends to decrease, but as shown by the broken line in Figure 9, the particle size at which sedimentation begins does not change depending on the dextran concentration and remains at 150. , increases as the iron ion concentration of the raw material aqueous solution increases.

The solubility of dextran in water is almost at its limit of 250 g/&. Also, as mentioned above, the iron ion concentration is 4
．． In order to prevent magnetite fine particles from settling under the conditions of 4 x I O-3moQ/(l, at least 50 g/C
A higher dextran concentration is required.

In order to obtain a solution with excellent dispersibility, as shown in Figure 6, a dextran concentration of 100 g/(! or more is required. Therefore, magnetite fine particles do not settle and an aqueous solution with good dispersibility is obtained. In order to achieve this, a dextran concentration of about 100 to 250 g/C is optimal.Also, in order to prevent magnetite fine particles from settling, the iron ion concentration in the raw material aqueous solution must be 6 × 10-'mo(/(l) or less (dextran concentration It can be seen that it is necessary to set it to 250g/(!).

(Example 5) Influenza viruses were labeled using dextran-coated magnetite microparticles produced by the production method of the present invention, collected by applying a magnetic field, and then observed using a transmission electron microscope (TEM). . The specific labeling method is as follows.

Dextran sugar chains are converted into periodate salts (e.g. Nal0).
*) to generate an aldehyde group, to which an IgG antibody obtained by purifying rabbit hyperimmune serum was bound. Figure 10 shows an electron micrograph of the thus obtained IgG antibody-dextran coated magnetite fine particles (
100,000 times). The dispersed dots that appear black are individual fine particles (magnetite appears as black dots), and it can be seen that they are very uniformly dispersed.

Next, add the influenza virus sample (antigen) to 35
The antigen-antibody reaction was performed by incubating at ℃ for 2.5 hours. Figure 11 shows an electron micrograph (1O
10,000 times). It can be seen that a large number of magnetically labeled antibodies (appearing black) are gathered and bound around several influenza virus particles with a diameter of approximately 1100 nm.

[Effects of the Invention] As described above, according to the method for producing magnetic fine particles of the present invention, magnetic fine particles whose particle size is controlled to 100 to 150 mm and excellent dispersibility in a solution can be obtained. be able to.

Further, in the production method of the present invention, by appropriately selecting the iron ion concentration and dextran concentration in the raw material aqueous solution, it is possible to control the particle size and the dispersibility of the obtained magnetic fine particles at the same time. Therefore, if the production method of the present invention is applied to the medical field, such as labeling materials in the field of immunological testing and carrier materials for drug delivery systems, it will be more useful by using magnetic properties and dispersion properties that could not be achieved with conventional magnetic fine particles. You can demonstrate your sexuality.

[Brief explanation of drawings]

Figure 1 is a graph showing the heating time of the raw material aqueous solution and the change in pH due to thermal decomposition of urea, and Figure 2 is a graph showing the relationship between the molar ratio of urea and iron ions in the raw material aqueous solution and the change in pH. The graph shown in Figure 3 is a schematic diagram showing a light scattering measurement system for determining the dispersion state of magnetic fine particles, and Figure 4 is a graph showing the Rayleigh ratio with respect to the scattering angle of magnetic fine particles.
The figure is a graph showing the results of measuring the particle size distribution of dextran-coated magnetite using a dynamic light scattering method. Figure 6 shows the change in Rayleigh ratio of the magnetic fine particle dispersion solution when the dextran concentration of the raw material aqueous solution is changed. Figure 7 is a graph showing the change in dispersibility of magnetic fine particles when the amount of urea added is changed, and Figure 8 is a graph showing the change in dispersibility of magnetic fine particles when the amount of urea added is changed. Magnetic particle X
A graph showing the line diffraction pattern, Figure 9 is a graph showing the relationship between the iron ion concentration in the raw material aqueous solution and the particle size of the obtained magnetic fine particles, and Figure 1O is an electron microscope of the IgG antibody-dextran coated magnetic fine particles. FIG. 11 is an electron micrograph of influenza virus bound to a magnetically labeled antibody.

Claims (1)

[Claims] Magnetic fine particles having an organic coating layer formed on their surfaces are produced by increasing the pH of a raw material aqueous solution containing divalent iron ions and/or trivalent iron ions and an organic coating layer forming material. A method in which the total iron ion concentration of the raw material aqueous solution is
ol/l or less, and by adding urea to a raw material aqueous solution and thermally decomposing the urea to increase the pH of the raw material aqueous solution, magnetite magnetic fine particles having a particle size of 150 Å or less are produced. manufacturing method.