# Patent

## Title
Ionic material

## Abstract

The present invention relates to a material containing ionic groups that form an ionic pair with a free radical catalyst. In particular, the invention relates to materials that are magnetic, and to the methods of forming such materials. For example, a method of forming paramagnetic particles is disclosed, the method comprising contacting a substance having cationic groups with an aqueous solution of Fe3+ so that all the cationic groups are exchanged for Fe3+, adjusting the pH of the solution to between 9 and 10, to form Fe(III) hydroxide, reducing a portion of the Fe(III) hydroxide to Fe(II) hydroxide to obtain a blend of the Fe(III) hydroxide the Fe(II) hydroxide, and crystallizing the two iron hydroxides to form paramagnetic particles comprising magnetite. The invention also discloses the formation of polymers from the ionic groups within the material. Various applications of the resulting materials are also disclosed.

## Background
This application is a divisional application of U.S. application Ser. No. 10/416,778 filed Sep. 10, 2003, now pending; which is a 35 USC § 371 National Stage application of PCT Application No. GB02/04677 filed Oct. 16, 2002; which claims the benefit under 35 USC § 119(a) of United Kingdom Application No. 0124812.9, filed Oct. 16, 2001. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.


The present invention relates to a material which contains ionic groups that are attached to the material, and form an ionic pair with a free radical catalyst. Polymers can then be formed from these site directed polymerisation points within the material. In addition the patent relates to a method for forming magnetic particles. The resulting materials can be used in various applications.
Non-magnetic and magnetic polymer particles are widely used in diagnostics, bioassays and life sciences research as they provide a means of separation of substances from complex mixtures. In brief, a ligand, e.g. antibody or antigen, is either non-covalently or covalently attached to the particle through chemical means. This complex is washed free of non-attached ligand and introduced into a sample e.g. blood containing a mixture of substances to compete for, or otherwise capture, analyte. The captured analyte is then detected using chemical, fluorescence or other means. The sensitivity and specificity of such separations depend, amongst other things, on the chemical properties of the particle.
It is a desired property of a microparticle that it contains chemical groups suitable for binding of a ligand and that the surface of the particle not reacted with ligand exhibit low non-specific binding characteristics.
The present invention provides a means to construct a particle with chemical groups attached to a polymer chain that are suitable for ligand binding. The polymer chain may be restricted to the interior of the particle and be accessible via the pores of the particle, or may extend to the surface of the particle or may extend beyond the surface of the particle.
The present invention also provides a means of manufacture of a superparamagnetic form of this particle by the deposition of magnetite at the ionic sites within the particle. A desired property of a superparamagnetic particle is that the concentration of the magnetite within the particle can be manipulated to achieve a suitably rapid response to a magnetic field. It is also a desirable property that the crystalline dispersion of the magnetite is fine enough such that each crystal cannot retain a residual magnetic moment after exposure to a magnetic field and that when the magnetic field is removed and the particles are re-dispersed they are not magnetically attracted to each other. The Japanese patent JP53146986 describes the addition of Fe2+ salts to a sulfonated polystyrene bead. The Fe2+ ions are then oxidized and precipitated to form magnetic particles. U.S. Pat. No. 4774265 describes the treatment of polymer particles with a solution of metallic ions capable of forming magnetic ferrites. The solution can swell or penetrate into the particles and as the particles contain groups capable of binding the metal salts, a proportion of the metal ions are retained. The metal hydroxides are precipitated by raising the pH, forming magnetic particles inside and on the surface of the particle. The present invention provides a method for forming superparamagnetic particles, by utilizing the ionic groups within the polymer particle for the capture Fe salts and then inducing nanocrystallization of magnetite. The ionic groups are released once the Fe is precipitated, allowing either the process of magnetite deposition to be repeated to control and increase the concentration of magnetite within the particles or to introduce free radical catalysts and subsequent polymer using site directed polymerization.
Addition polymers are formed by the activation of a free radical catalyst in the presence of suitable monomer(s). The position of the catalyst in a reaction is normally random so the location of the polymer is imprecise. The present invention provides a material which comprises one or more ionic groups which have formed ionic pairs with one or more free radical catalysts. Polymers will be formed at the site of the catalyst. By controlling the position of the ionic groups and the conditions of the polymerisation the position of the formed polymer can be directed and hence the architecture of the final composite controlled.
Suitable ionic groups include sulfates, sulfonates, phosphates, carboxylates, phenolics, siloxane, primary, secondary, tertiary or quaternary amines. A preferred material is a strong anion exchange resin such as a sulfonated polystyrene resin or a strong cation exchange resin.
The material can be polymeric in nature such as sulfonated or aminated polystyrene or poly(meth)acrylate, or may be non-polymeric such as silica or mica. The shape of the particle is normally spherical, e.g. beads, but may also be in the form of a membrane, mesh, film, gel, plug or any other type suitable to bioassay reagent formulations.
The free radical catalyst is ionic in nature and capable of forming an ion pair species with the ion containing material. The catalyst may be capable of thermal or redox initiation. Preferred thermal catalysts include 2,2′-azobis (2 amidinopropane) dihydrochloride, 2,2′-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride, 4,4′-Azobis(4-cyanopentanoic acid), ammonium persulfate or sodium persulfate. Preferred redox catalyst is Iron III chloride used in conjunction with hydrogen peroxide or other peroxide catalysts.
Other free radical catalysts capable of forming an ion pair species with the ion containing material can be used.
The material of the invention is formed by a free radical catalyst being absorbed via an ion exchange mechanism into the material, as the material contains ionisable groups which allow this exchange to occur.
The material is washed with a solution of the catalyst so that the catalyst is absorbed. The catalyst should be an organic or inorganic salt carrying an opposite charge to the charge of the ion groups within the material. An ion exchange equilibrium occurs in which the catalyst forms an ion pair with the ionic group attached to the material.
Subsequent washing with a non-ionic solution removes excess unbound catalyst but is unable to remove the ion-paired catalyst.
In a second aspect the present invention provides a method of forming a composite material comprising of the following steps:

    
    
        (a) Addition of one or more monomers to material of the present invention;
        (b) Generation of free radicals by the catalyst
        (c) Formation of a polymer from the catalyst attached to the ionic groups.
    
    


The polymers can either extend beyond the outer surface of the material to form short spacers or long extensions of several microns in length, or remain wholly within the material. The polymers are produced via a free radical mechanism and are typically styrenic, (meth)acrylate, (meth)acrylamides, vinyl acetate. Any monomers polymerisable via a free radical mechanism could be used.
Suitably the catalyst-loaded material is added to a solution or suspension containing monomers, and exposed to heat or a co-catalyst. The catalysts decompose to generate free radicals. Therefore polymerisation occurs at the site of the catalyst, which is held by ionic interactions within the material.
A composite material so formed represents a third aspect of the invention.
In a fourth aspect the present invention provides a method of preparing magnetic material comprising of the following steps:

    
    
        (a) Contacting a material comprising cationic groups with a solution of Fe3+ so that all the cationic sites are exchanged for Fe3+
        (b) Adjusting the pH to pH 9-10, e.g. by using ammonia to precipitate Fe(III)hydroxide
        (c) Reducing at least some of the Fe(III) hydroxide to Fe(II) hydroxide
        (d) Crystallization of the two iron hydroxides to form magnetite (Fe3O4).

Those having ordinary skill in the art would know how to design an appropriate protocol for a process of crystallization in step (d) to obtain magnetite having optimal properties. For example, a mixture of the two iron hydroxides can be filtered and washed using deionized water until the eluent is clear.

    
    


The cationic groups are reformed at the end of step d, allowing the process of Fe3+ addition to be repeated to increase the concentration of magnetic particles within the material before the polymer is generated. Indeed, during the process of precipitation, when the Fe3+ ions are exposed to the environment having the degree of basicity described in step (b), the cationic groups are regenerated. The reduction step (c) can suitably be achieved using hydrazine.
The method for forming the paramagnetic material can further comprise the following steps:

    
    
        (e) Loading the reformed cationic groups with free radical catalyst
        (f) Addition of one or more monomers to material;
        (g) Generation of free radicals by the catalyst
        (h) Formation of a polymer from the catalyst attached to the ionic groups, in order to form a paramagnetic composite material.
    
    


The oxidation process described in Japanese patent JP53146986 can also be employed to introduce the magnetite or a ferrite using Fe2+ or a mixture of Fe2+ and other metal2+ ions such as Ni2+, Mn2+ prior to the formation of the composite particles. Thus another aspect of the invention provides a method of forming a paramagnetic material comprising of the following steps:

    
    
        (a) Contacting a material having cationic groups with a solution containing Fe2+ ions so that all the cationic sites are exchanged for a metal2+ ion;
        (b) Adjusting the pH to pH9-10;
        (c) Aerating the material at 65° C. until it gains magneticity
        (d) Loading the reformed cationic groups with free radical catalyst
        (e) Addition of one or more monomers to material;
        (f) Generation of free radicals by the catalyst
        (g) Formation of a polymer from the catalyst attached to the ionic groups.
    
    


The material of the present invention can then be used in diagnostic assays such as bioassays, immunoassays, flow cytometry, and DNA hybridisation. For instance, polymeric extensions formed according to the methods of the invention can be used for the attachment of other bioassay particles such as immunoglobulins.



The invention will now be described with reference to the following examples, which should in no way be construed as limiting the invention. The Examples refer to the following figures:
FIG. 1 Optical micrograph of beads with polyacrylic acid tentacle structure.
FIG. 2 Optical micrograph of magnetic beads with a poly(vinyl acetate) layer around the surface of the beads.



EXAMPLES
1) Magnetic Particles
a) Magnetite Loading:
30 g of a sulfonated polystyrene beads (8 μm modal size, 8% cross-linked) with an hydrogen counter-ion was washed on a sinter with 1L of a 5 wt % iron III chloride solution. The beads were then washed with R.O. water until the eluent was free of Fe3+ ions. The loaded beads were dispersed into R.O. water and made up to a total slurry weight of 550 g. This slurry was poured into a 1L, 3 necked round bottom flask fitted with a condenser, centrifugal stirrer and nitrogen inlet. The pH was adjusted to 9.5 with ammonia solution and the slurry was purged with nitrogen for 1 hour at 85° C. 3 mls of a 35 wt % solution of hydrazine was added and the reaction allowed to proceed with gentle stirring for 3 hours. A colour change for the slurry from orange-brown to brown-black was noted.
The reaction was cooled and the beads filtered on a sinter and washed with R.O. water. A small amount of black particles were washed from the beads. Washing was continued until the eluent was clear.
The beads were contacted with iron III chloride solution and reduced with hydrazine two further times to produce composite superparamagnetic particles with a magnetic susceptibility value of 59.7 m3/Kg.
b) Initiator Loading:
1 g of magnetic particles from section (a) were washed on a sinter with 50 mls of a 2 wt % solution of 2,2′ azobis(2-methylpropionamidine) dihydrochloride in R.O. water. The beads were then washed with R.O. water until the eluent was free of chloride ions. The beads were then titrated on a Metrohm autotitrator against standardised potassium hydroxide to determine the free sulfonic acid groups remaining in the beads, 1 g of magnetic particles of example 1 were also titrated before contact with initiator. The results are in table 1:









TABLE 1







free —SO3H (mmol/g)



















magnetic particles from example 1
2.06



initiator loaded magnetic
0.15



particles from example 1










Results indicate that  ̃1 mmol of initiator was loaded per g of magnetic beads.

c) Site Specific Initiation:

1 g of initiator loaded beads from section (b) was dispersed into 30 g of R.O. water/1 g acrylamide monomer in a 3 necked round bottom flask fitted with a condensor, centrifugal stirrer and nitrogen inlet. The suspension was purged with nitrogen for 20 mins and the temperature then increased to 70° C. for 2 hrs.
The product was filtered and washed with water. The beads were then dispersed in 1M NaOH solution at 60° C. for 1 hour to hydrolise the acrylamide to acrylic acid. Resultant beads have a tentacle structure with the acrylic acid stretching away from the surface. An optical micrograph of the beads is shown in FIG. 1.
2) Poly(vinyl acetate) Coated Particles:
1 g of initiator loaded particles from example 1 were dispersed in 75 ml of water containing 3.8 g of ALCOTEX 78 which is a poly(vinyl alcohol)-based suspending agent available from Harlow Chemicals. This suspension was poured into a 250 ml round bottomed flask fitted with a condenser, nitrogen inlet and centrifugal stirrer. 8.3 g of vinyl acetate monomer was added to the suspension and the mixture stirred and purged with nitrogen for 2 minutes. The temperature was increased to 55° C. and polymerization continued for 24 hours. The resultant beads were filtered, and washed with water. Optical microscopy (FIG. 2) indicated a visible poly(vinyl acetate) layer about the surface of the magnetic beads.

## Claims

1. A method of preparing superparamagnetic particles attached to a polymer chain comprising the steps of:
(a) providing particles comprising ionic groups;
(b) utilizing the ionic groups for the capture of Fe cations;
(c) inducing nanocrystallization of magnetite or ferrite to form superparamagnetic particles; and
(d) introducing free radical catalysts and subsequent polymer using site directed polymerization.

2. The method of claim 1, wherein the particles in step (a) are polymeric or non-polymeric.

3. The method of claim 1, wherein the ionic groups are selected from sulfates, sulfonates, phosphates, carboxylates, or phenolics.

4. The method of claim 1, wherein the particles in step (a) comprising ionic groups are selected from sulfonated polystyrene or poly(meth)acrylate particles.

5. The method of claim 1, wherein step (b) comprises:
contacting the particles comprising ionic groups with an aqueous solution Fe3+ or Fe2+ so that all the cations are exchanged for an Fe3+ or an Fe2+ ion.

6. The method of claim 5, wherein step (c) comprises:
adjusting the pH of the solution of Fe3+ to between 9 and 10 to precipitate Fe(III) hydroxide;
reducing at least some of the Fe(III) hydroxide to Fe(II) hydroxide; and
crystallizing the two iron hydroxides to form magnetite (Fe3O4).

7. The method of claim 5, wherein step (c) comprises:
adjusting the pH of the solution of Fe2+ to between 9 and 10; and
oxidizing the Fe2− ions to form paramagnetic particles by aerating the particles at 65° C.

8. The method of claim 1, wherein the steps (b)-(c) are carried out a plurality of times.

9. The method of claim 6, wherein said reducing is carried out using hydrazine.

10. The method of claim 5, wherein the solution containing Fe2+ ions further comprises additional metal 2−ions.

11. The method of claim 10, wherein the additional metal 2+ ions are Ni2+ or Mn2+ or a combination thereof.

12. The method of claim 1, wherein step (d) comprises the following steps:
loading the ionic groups with the free radical catalyst;
adding one or more monomers to the particles;
generating free radicals by the catalyst; and
forming a polymer from the catalyst attached to the ionic groups.

13. The method of claim 12, wherein the free radical catalyst is capable of thermal or redox initiation.

14. The method of claim 13, wherein the free radical catalyst capable of thermal initiation is selected from 2,2′-azobis (2 amidinopropane) dihydrochloride, 2,2′-azobis [2-(2-imidazolin-2-yl) propane) dihydrochloride, 4,4′-azobis (4-cyanopentanoic acid), ammonium persulfate or sodium persulfate.

15. The method of claim 13, wherein the free radical catalyst capable of redox initiation is Iron III in conjunction with a peroxide catalyst.

16. The method of claim 1, wherein the polymer is selected from styrene, (meth)acrylate, (meth)acrylamide, or vinyl acetate.

17. The method of claim 1, wherein the polymer chain further comprises chemical groups suitable for ligand binding.