U.S. patent application number 13/266910 was filed with the patent office on 2012-06-07 for monodisperse submicron polymer particles.
This patent application is currently assigned to INVITROGEN DYNAL AS. Invention is credited to Geir Fonnum, Nini Kjus, Silje Lien, Grete Irene Modahl, Astrid Molteberg.
Application Number | 20120141798 13/266910 |
Document ID | / |
Family ID | 40791997 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141798 |
Kind Code |
A1 |
Modahl; Grete Irene ; et
al. |
June 7, 2012 |
MONODISPERSE SUBMICRON POLYMER PARTICLES
Abstract
This invention relates to monodisperse cross-linked polymer
particles, comprising particles with a substantially smooth outer
surface and an average diameter of less than 1 .mu.m, wherein the
particles are solid or porous, and wherein the coefficient of
variation (CV) % of the particles, when measured by CPS disk
centrifugation analysis, is less than 15%. These monodisperse
cross-linked polymer particles may comprise magnetic material and
are useful in various application. This invention also relates to
monodisperse polymer particles for use as seed particles in the
Ugelstad process.
Inventors: |
Modahl; Grete Irene;
(Smedstad, NO) ; Fonnum; Geir; (Smestad, NO)
; Molteberg; Astrid; (Smestad, NO) ; Lien;
Silje; (Smestad, NO) ; Kjus; Nini; (Smestad,
NO) |
Assignee: |
INVITROGEN DYNAL AS
Carlsbad
CA
|
Family ID: |
40791997 |
Appl. No.: |
13/266910 |
Filed: |
April 29, 2010 |
PCT Filed: |
April 29, 2010 |
PCT NO: |
PCT/EP2010/055874 |
371 Date: |
February 22, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61174407 |
Apr 30, 2009 |
|
|
|
Current U.S.
Class: |
428/407 ;
428/402; 428/403; 526/181 |
Current CPC
Class: |
C08F 257/02 20130101;
C08F 257/02 20130101; C08F 257/02 20130101; C08J 2325/08 20130101;
C08J 3/128 20130101; C08F 12/08 20130101; C08F 212/36 20130101;
C08F 8/30 20130101; C08F 212/36 20130101; C12N 11/08 20130101; C08F
8/30 20130101; C08F 12/08 20130101; C08J 9/20 20130101; Y10T
428/2998 20150115; C08J 9/18 20130101; Y10T 428/2991 20150115; Y10T
428/2982 20150115; C08F 2/22 20130101; C08F 212/36 20130101; C08F
2/22 20130101; C08F 212/08 20130101; C08F 257/02 20130101; C08F
2800/20 20130101; C08F 2/22 20130101 |
Class at
Publication: |
428/407 ;
526/181; 428/402; 428/403 |
International
Class: |
B32B 15/02 20060101
B32B015/02; B32B 5/16 20060101 B32B005/16; C08F 4/46 20060101
C08F004/46 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 29, 2009 |
GB |
0907372.7 |
Claims
1. Monodisperse cross-linked polymer particles having the following
characteristics: a substantially smooth outer surface a z-average
diameter of less than 1 .mu.m the particles are porous a
coefficient of variation (CV) %, when measured by CPS disk
centrifugation analysis, of less than 15%.
2. The polymer particles of claim 1, wherein the polymer particles
have a z-average diameter of no more than 900 nm, optionally no
more than 700 nm, further optionally no more than 600 nm, still
further optionally no more than 500 nm.
3. The polymer particles of claim 1 or claim 2, wherein the polymer
particles have a z-average diameter of at least 200 nm, optionally
at least 300 nm, e.g. at least 400 nm.
4. The polymer particles of any preceding claim, wherein the
coefficient of variation (CV) % of the particles, when measured by
CPS disc centrifugation analysis, is less than 12%, optionally less
than 10%, further optionally less than 5%.
5. The polymer particles of any preceding claim, wherein the
polymer particles constitute a first population of particles and
have a swellability of not more than the swellability of a second
population of polymer particles, wherein the second population of
particles are reference particles produced by a known Ugelstad
process and made of a comparable polymer to the first population,
wherein the amount of cross-linker monomer used in the suspension
polymerisation stage of the known Ugelstad process is >25% by
weight of the total weight of monomers used in the suspension
polymerisation stage, optionally wherein the first population of
polymer particles are polystyrenic particles and the monomers used
in the suspension polymerisation stage in the preparation of the
reference particles were styrene and, as a crosslinker monomer,
divinylbenzene, the divinylbenzene being in an amount of >25% by
weight of the total of styrene plus divinylbenzene.
6. The polymer particles of claim 5, wherein the amount of the
cross-linker monomer used in said suspension polymerisation stage
is 30-60%, optionally 40-50%.
7. The polymer particles of any preceding claim, wherein the
particles have a specific surface area of from 300 to 700 m.sup.2/g
when measured by gas adsorption analysis.
8. (canceled)
9. The polymer particles of any of claims 1 to 6, further
comprising a magnetic material, optionally wherein the magnetic
material is a metal oxide or alloy e.g. ferrimagnetic or
superparamagnetic iron oxide crystals, further optionally wherein
the magnetic material is substantially located in pores of the
polymeric material.
10. The polymer particles of any of claims 1 to 6, further
comprising a superparamagnetic material, optionally wherein the
superparamagnetic material is a metal oxide or alloy, further
optionally wherein the superparamagnetic material is substantially
located in pores of the polymeric material.
11. The polymer particles of claim 10, wherein the
superparamagnetic material comprises a ferrite or coated iron based
or iron-nickel alloy nanoparticles, optionally wherein the
superparamagnetic material comprises a ferrite selected from
magnetite, maghemite, or magnetite or maghemite further optionally
where the iron is substituted with up to 5 mol % of Al, Mn, Ni, Cu,
Co, Zn, Ca, Ge, Te, Ti or Sn, further optionally wherein the
superparamagnetic material comprises magnetite or maghemite in
either case substituted with up to 5 mol % manganese.
12. The polymer particles of any preceding claim, wherein the outer
surface further comprises a coating and/or a functional group,
optionally wherein the functional group is selected from carboxylic
acid, amine, tosyl, epoxy and hydroxyl.
13. The polymer particles of any preceding claim, wherein the
polymer comprises a cross-linked polymer selected from polymers and
copolymers of one or more of the following monomeric materials: an
acrylic monomer, e.g. methacrylate, and a styrenic monomer,
optionally wherein the polymer is polystyrene.
14. The polymer particles of claim 10 or claim 11 which comprise: a
matrix polymer which is selected from polymers of styrenic monomers
and polymers of acrylic monomers and optionally is a polystyrene;
magnetic crystals; a polymer coating which is optionally formed
from at least one epoxide compound.
15-23. (canceled)
24. Monodisperse particles of any preceding claim which have a
silica coating.
25. Monodisperse particles of any preceding claim, wherein the
polymer particles do not comprise methylene bridged phenyl
groups.
26-32. (canceled)
33. A process for the preparation of monodisperse particles for use
as Ugelstad seed particles, the process comprising an emulsion
polymerisation process comprising: forming an aqueous dispersion
comprising a vinylic monomer and a water soluble polymerisation
initiator; and agitating until the commencement of particle
nucleation; characterised in that: the aqueous dispersion comprises
a surfactant and a chain transfer agent is added after the
commencement of particle nucleation, such that the polymerisation
forms monodisperse seed particles having a z-average diameter of
from 50 nm to 200 nm and, when measured by gel permeation
chromatography, the polymer has a mean weight average molecular
weight of more than 1,000 and less than 70,000.
34. The process of claim 33, wherein the surfactant is present
below its critical micelle concentration.
35. The process of claim 33, wherein the surfactant is present in a
concentration of not more than 2.5 g/L, optionally not more than 2
g/L, further optionally not more than 1.7 g/L, still further
optionally not more than 1.5 g/L.
36. (canceled)
37. The process of any of claims 33 to 35, wherein the surfactant
is sodium dodecyl sulfate.
38. (canceled)
39. The process of any of claims 33 to 37 or claim 37, wherein the
vinylic monomer is an acrylic monomer, a styrene monomer or a
methacrylate monomer, optionally wherein the monomer comprises
styrene.
40. The process of any of claims 33 to 35, or claim 37 or claim 39,
wherein the chain transfer agent comprises a haloalkane, optionally
bromotrichloromethane.
41. The process of any of claims 33 to 35 or claim 37 or claims 39
to 40, wherein the chain transfer agent comprises an alkymercaptan,
optionally butyl mercaptan, isooctyl 3-mercaptopropionate or
octylmercaptan.
42. The process of any of claims 33 to 35 or claim 37 or claims 39
to 41, wherein the addition of chain transfer agent starts about 5
to 15 minutes after the commencement of visible particle
nucleation, optionally about 10 minutes after the commencement of
visible particle nucleation.
43-46. (canceled)
47. The process of any of claims 33 to 35 or claim 37 or claims 39
to 42, further comprising subjecting the monodisperse seed
particles to an Ugelstad process wherein the percentage by weight
of cross-linker monomer included in the total monomer used in the
suspension polymerisation stage of the Ugelstad process (the final
polymerisation stage where there are plural polymerisation stages)
is >25% wt cross-linker, to form submicron monodisperse
particles and a z-average diameter of less than 1 .mu.m, optionally
wherein the submicron monodisperse particles are porous particles
having a smooth outer surface, e.g. wherein the submicron
monodisperse particles have a specific surface area of from 300 to
700 m.sup.2/g when measured by gas adsorption analysis.
48. The process of claim 47, wherein the Ugelstad process
comprises: (i) forming an aqueous dispersion comprising the
monodisperse seed particles, finely divided droplets comprising an
organic compound of molecular weight below 5,000 and water
solubility at 25.degree. C. of less than 10.sup.-2 g/L, and an
organic solvent in which the organic compound is soluble, the
organic solvent being optional when the polymer forming the seed
particles has an average molecular weight which corresponds to up
to 50 monomer units; (ii) allowing the organic compound to diffuse
into the monodisperse seed particles, causing the seed particles to
become activated; (iii) removing the organic solvent, where present
from inside the seed particles, and contacting the activated seed
particle with an aqueous vehicle containing a monomer that is at
least 10 times more soluble in water than the organic compound, and
a crosslinker; (iv) allowing the monomer to diffuse into the
activated seed particles to form an aqueous dispersion of swollen
seed particles; and (v) initiating polymerisation of the monomer in
the swollen seed particles.
49-70. (canceled)
Description
[0001] This invention relates to monodisperse polymer particles
useful in biological assays and other applications. It also relates
to processes for preparing such particles, intermediates used in
such processes and methods of using the particles, as well as other
subject matter.
BACKGROUND
[0002] A common reaction for making polymers is free radical
polymerisation, which is used to make polymers from unsaturated
monomers, for example styrene and/or acrylates. Free radical
polymerisation may be performed by emulsion polymerisation or
suspension polymerisation. Emulsion polymerisation is the standard
process for production of polymer particles in sizes around 100-500
nm. The product is often called a latex and is the main component
of water based paints. In emulsion polymerisation unsaturated
monomers are, as just mentioned, converted to polymers by the use
of free radical polymerisation. Typically the polymerisation is
carried out in water and the monomers have a low water
solubility.
[0003] In a typical procedure, a monomer such as styrene, for
example, is mixed with water and surfactants and the mix is stirred
to make relatively large styrene droplets (1-10 .mu.m). A water
soluble initiator is added. The initiator decomposes to two
radicals and starts reacting with the monomers that have been
solubilised in the water phase. In the case of styrene, the growing
chain soon becomes water insoluble and the molecules aggregate to
nanometer-sized particles. If the initiation phase is short the
resulting particles can be monosized.
[0004] The particles then grow by the following mechanism: The
monomers diffuse through the water phase into the polymer
particles. The polymerisation in the particles is initiated by the
adsorption of growing polymers from the water phase. Since the
particles are so small only one radical can survive in the particle
at the same time, and the adsorption of a second growing chain will
therefore result in termination. The polymerisation stops when the
monomer in the large droplets has diffused into the growing
particles and polymerised.
[0005] Emulsion polymerisation is dependent on the transfer of
monomer from the large droplets to the smaller particles and
radical adsorption. If a high amount of crosslinking monomer is
used--that is above 10-15%--the small particles will be crosslinked
and the polymerisation does not continue.
[0006] Emulsion polymerisation is used to produce monosized seed
particles as starting material for the Ugelstad two step swelling
process.
[0007] This application teaches a method which is able to produce
submicron highly crosslinked polymer particles which it is believed
cannot be obtained by emulsion polymerisation. It is known to
produce cross-linked porous or solid monodisperse polymer particles
by a two stage process, named the Ugelstad process after the late
Professor John Ugelstad, which is described for example in
EP-B-3905 (Sintef) and U.S. Pat. No. 4,530,956 (Ugelstad). An
improved Ugelstad process is described in WO 00/61647. In the
Ugelstad process, seed particles, suitably made by emulsion
polymerisation, are converted in two steps into monodisperse
particles by seeded suspension polymerisation. In a first step, the
seed particles are swollen by making a fine (e.g. submicron)
aqueous emulsion of a relatively low molecular weight
water-insoluble substance and then adding a water-miscible organic
solvent (e.g. acetone) so that the water-insoluble substance
diffuses into the seed particles. It is convenient for the
water-insoluble substance to be a heat-activated polymerisation
initiator. In a second step, the solvent is then removed, locking
the water-insoluble substance in the seed particles, and the seed
particles take up a large quantity of monomer and also a
cross-linker, driven by an increase in entropy when the monomer and
cross-linker diffuse into the seed particles and dilutes the
water-insoluble substance. In practice, after the seed particles
have been swollen and absorbed the water-insoluble substance, the
dispersion containing them is typically contacted with an aqueous
emulsion containing the monomer and cross-linker; the amount of
water is chosen to be sufficient for the water to act to remove the
water-miscible solvent by dilution and the monomer is driven into
the seed particles. The seed particles swell and, following
initiation of polymerisation, e.g. by heating to activate the
initiator, larger polymer particles are produced. The Ugelstad
process therefore comprises making seed particles by emulsion
polymerisation and expanding the seed particles by suspension
polymerisation. The smallest monodisperse particles described in
the aforementioned prior art have an average diameter of 1
.mu.m.
[0008] A porogen may be contacted with the seed particles in the
final swelling and polymerisation stage in order to make the
particles porous. A porogen is an organic liquid which does not
participate in the polymerisation reaction. It may be a good
solvent for the polymer in which case it will make small pores, or
a poor solvent for the polymer, in which case it will form large
pores. The present inventors have not been able to control the pore
morphology of submicron beads to provide a smooth outer surface by
selection of porogens.
[0009] In a simplified version of the Ugelstad process, the
enhanced capacity for swelling may be achieved simply by the use of
oligomeric seed particles, e.g. where the oligomer weight average
molecular weight corresponds to up to 50 monomer units (a molecular
weight of about 5000 in the case of polystyrene).
[0010] Conveniently, a very fine submicron (e.g. 0.05-0.5 .mu.m)
stable oil-in-water emulsion can be made if there is used as the
emulsifier a combination of a water-soluble surfactant and a less
water soluble organic compound. The surfactant is usually ionic but
alternatively may be non-ionic.
[0011] Prior art Ugelstad methods described above do not provide
smooth monodisperse porous particles at small particle sizes. In
particular, the prior art methods do not produce smooth particles
at the lower size limits. Instead, the outer surface of these small
porous particles would be irregular and, for example appear rough
or knobbly when viewed at a magnification of e.g. 10,000, e.g. when
compared to larger particles. This rough appearance is caused by
the morphology of the pores, e.g. excessive pore size.
BRIEF SUMMARY OF THE DISCLOSURE
[0012] The invention is in part based on an appreciation that
smaller particles than previously made by the Ugelstad process
would be expected to increase the sensitivity and reproducibility
of assays using them, as compared to larger particles (on a per
gram basis). For example when compared to particles of greater than
1 .mu.m diameter submicron particles provide many more particles
per gram (FIG. 1), leading to increased surface area and better
statistics for small samples. Smaller particles would significantly
reduce the amount of biological material required for a biological
assay because less volume will be occupied by the particle itself.
In addition, submicron particles have application in micro- and
nano-fluidics and other nanotechnology areas.
[0013] Polymer particles may have an irregular shape, with a
surface which appears rough or knobbly under magnification, for
instance when viewed under a magnification of 10,000 with a
scanning electron microscope. It has further been appreciated that
for some applications it would be beneficial to provide a technique
for preparing particles having a surface which gives an overall
smooth appearance to the particles; such particles have an
appearance more of regular spheres than irregular shapes.
[0014] It has thus been appreciated that smooth polymer particles
have advantages when compared to polymer particles with an
irregular shape. One advantage is that where the particles are
magnetic, smooth particles provide a uniform magnetic signal in all
directions, unlike irregular particles. This provides for a more
consistent performance when exposed to a magnetic field. A further
advantage of smooth particles is that all of the outer surface is
readily accessible, e.g. to a solution. This advantage is
particularly important where the outer surface is functionalised,
for example with antibodies. Antibodies or other ligands attached
to any part of the surface of a smooth particle are able to
interact with binding partners present in a solution, potentially
providing a sensitive and reproducible assay. Antibodies or other
ligands attached to the surface of a rough polymer particle can be
located at sites with limited access to the solution, reducing the
sensitivity and/or reproducibility of the assay.
[0015] The present invention is believed to enrich the field of
polymer particles and the technology of their manufacture in a
number of ways. Thus, the invention provides novel monodisperse
submicron polymer particles having characteristics which it is
believed cannot be obtained repeatedly, if at all, by emulsion
polymerisation; such characteristics may include one, two, three,
four or five of: high cross-linking; porosity; morphology, swelling
characteristics, and magnetism (which may be implemented by the
incorporation of magnetic particles). The invention includes within
its ambit monodisperse submicron polymer particles having
characteristics which it is believed could not previously be
obtained by Ugelstad polymerisation; such characteristics may
include one of more of morphology (in particular porous particles
which appear smooth or spherical to the eye when viewed at a
scanning electron microscope magnification of 10,000) and size. It
will be appreciated therefore that certain implementations of the
invention embrace monodisperse submicron particles having
characteristics which it is believed cannot be obtained by previous
processes for making polymer particles.
[0016] The invention also enriches polymer particle technology with
a variant of the Ugelstad process which is capable of making the
polymer particles described herein. The invention also provides a
novel seed particle useful in the Ugelstad process to control the
pore size and hence surface smoothness of the end product
particles. In this regard, aspects of the invention are predicated
on a finding that the pore size of the end product particles may be
controlled by controlling the size of the polymer molecules forming
the seed particle.
[0017] The products, processes and uses of the invention are not
limited to the subject matter just-mentioned but are, without
limitation, described more fully in the following description and
claims and illustrated by the accompanying drawings.
[0018] In accordance with one aspect of the present invention there
are provided monodisperse, cross-linked submicron polymer
particles, i.e. monodisperse particles having an average diameter
of less than 1 .mu.m. ("Submicron" means less than 1 .mu.m.)
[0019] The particles may be magnetic. The invention includes
monodisperse magnetic polymer particles comprising a matrix polymer
and magnetic particles, the particles having an average diameter of
less than 1 .mu.m. In one embodiment, the magnetic particles are
superparamagnetic crystals and in another embodiment they are
ferromagnetic, e.g. the magnetic particles may be ferrimagnetic or
superparamagnetic iron oxide crystals.
[0020] Another aspect of the invention resides in monodisperse,
cross-linked polymer particles having a smooth outer surface.
[0021] The invention includes monodisperse, cross-linked submicron
polymer particles having a smooth outer surface.
[0022] As explained in more detail below, "monodisperse" refers
herein to particles having a low coefficient of variation (CV) of a
specific parameter (e.g. particle diameter), for example a CV of
less than 20% and is particular of less than 15%, e.g. of less than
10% and sometimes of less than 5%.
[0023] The invention includes within its scope both porous and
solid particles. So-called "solid" particles may have very low
porosity, as is known in the art, and are alternatively called
"compact" particles.
[0024] One class of submicron particles disclosed herein are
monodisperse and porous. These particles may have specified surface
characteristics. In one sub-class the surface is smooth. In another
sub-class, the specific surface area of the particles is from 300
m.sup.2/g to 700 m.sup.2/g, when measured by gas absorption
analysis.
[0025] Further included in the invention are monodisperse polymer
particles useful as seed particles for making the described
submicron particles, wherein the seed particles have an average
diameter of from 50 nm to 200 nm and wherein the polymer has a mean
weight average molecular weight of more than 1,000 and less than
70,000, when measured by gel permeation chromatography. In
embodiments the average diameter is from 50 nm to less than 200
nm.
[0026] In another aspect, there is provided a process for the
preparation of monodisperse polymer particles for use as seed
particles in seeded suspension polymerisation, the process
comprising an emulsion polymerisation process comprising:
[0027] forming an aqueous dispersion comprising an ethylenically
unsaturated monomer and a water soluble polymerisation initiator;
and
[0028] mixing until the commencement of particle nucleation;
characterised in that:
[0029] the aqueous dispersion comprises a surfactant and in that a
chain transfer agent is added after the commencement of particle
nucleation, such that the polymerisation forms monodisperse seed
particles having an average diameter of from 50 nm to 200 nm and,
when measured by gel permeation chromatography, the polymer has a
mean weight average molecular weight of more than 1,000 and less
than 70,000. In embodiments, the average diameter of the seed
particles is from 50 nm to less than 200 nm.
[0030] The invention includes the use of the mentioned monodisperse
seed particles as seed particles in an Ugelstad process. In this
way, the seed particles may be used as an intermediate in the
preparation of submicron and/or smooth polymer particles described
herein.
[0031] The Ugelstad process may comprise: [0032] (i) forming an
aqueous dispersion comprising [0033] the monodisperse seed
particles, [0034] finely divided droplets comprising an organic
compound of molecular weight below 5,000 and water solubility at
25.degree. C. of less than 10.sup.-2 g/L, and [0035] an organic
solvent in which the organic compound is soluble, the organic
solvent being optional when the polymer forming the seed particles
has an average molecular weight which corresponds to up to 50
monomer units; [0036] (ii) allowing the organic compound to diffuse
into the monodisperse seed particles, causing the seed particles to
become activated; [0037] (iii) removing the organic solvent, where
present, from inside the seed particles, and contacting the
activated seed particle with an aqueous vehicle containing a
monomer, for example one that is at least 10 times more soluble in
water than the organic compound, and a crosslinker; [0038] (iv)
allowing the monomer to diffuse into the activated seed particles
to form an aqueous dispersion of swollen seed particles; and [0039]
(v) initiating polymerisation of the monomer in the swollen seed
particles.
[0040] The invention includes particles which have been obtained by
the processes described in this specification.
[0041] The invention also includes particles having the
characteristics of particles obtained by the methods disclosed
herein; whilst such particles are obtainable by the processes
described herein, they are characterized solely by their properties
and not by their method of manufacture and, accordingly, the scope
of protection of claims directed to particles specified by their
characteristics is determined solely by the characteristics of the
particles to the exclusion of their actual method of
manufacture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] Embodiments of the invention are further described
hereinafter with reference to the accompanying drawings, in
which:
[0043] FIG. 1 is a graph indicating the relationship between bead
number per gram and diameter for beads of the disclosure having a
particle density of 1.5 g/cm.sup.3;
[0044] FIG. 2 provides an overview of an Ugelstad process;
[0045] FIG. 3 illustrates the diameter and coefficient of variation
(CV %) of the diameter obtained by disc centrifugation for
polystyrene seed particles formed according to the procedure of
Example 1, with a weight average CV of the main peak of 4% as
determined from the diameter range of 0.1-0.17 .mu.m;
[0046] FIGS. 4 to 6 provide SEM images of submicron polymer
particles, formed by application of an Ugelstad process to seed
particles comprising low molecular weight polymer;
[0047] FIG. 7 is a graph illustrating how seed particle diameter is
affected by sodium dodecyl sulfate (SDS) concentration, both with
(diamond data points) and without (square data points) the presence
of a chain transfer reagent;
[0048] FIG. 8 is a graph illustrating the molecular weight
distribution of various polystyrene seed particles, as measured by
gel permeation chromatography (GPC);
[0049] FIG. 9 illustrates the diameter and coefficient of variation
(CV %) of the diameter obtained by disc centrifugation for
polystyrene seed particle L1740, with a weight average CV of main
peak of 2% as determined from the diameter range of 0.15-0.50
.mu.m;
[0050] FIGS. 10A to 10D are micrographs of four different submicron
porous polymer particles;
[0051] FIG. 11 illustrates the diameter and coefficient of
variation (CV %) of the diameter obtained by disc centrifugation
for porous polystyrene particle LK321, with a weight average CV of
main peak of 8% as determined from the diameter range (0.27
.mu.m-0.7 .mu.m);
[0052] FIG. 12 illustrates the diameter and coefficient of
variation (CV %) of the diameter obtained by disc centrifugation
for polystyrene polymer particle L1713, with a weight average CV of
main peak of 2% as determined from the diameter range (0.4
.mu.m-0.6 .mu.m);
[0053] FIG. 13 is an SEM of prior art MyOne.TM. polymer
particles;
[0054] FIG. 14 is (a) an SEM and (b) a TEM of commercial sample
1;
[0055] FIG. 15 is (a) an SEM and (b) a TEM of commercial sample
2;
[0056] FIG. 16 is (a) an SEM and (b) a TEM of commercial sample
3;
[0057] FIG. 17 is (a) an SEM and (b) a TEM of commercial sample
4;
[0058] FIG. 18 is (a) an SEM and (b) a TEM of commercial sample
5;
[0059] FIG. 19 is (a) an SEM and (b) a TEM of commercial sample
6;
[0060] FIG. 20 is (a) an SEM and (b) a TEM of commercial sample
7;
[0061] FIG. 21 is (a) an SEM and (b) a TEM of commercial sample
8;
[0062] FIG. 22 is (a) an SEM and (b) a TEM of commercial sample
9;
[0063] FIG. 23 is (a) an SEM and (b) a TEM of commercial sample
10;
[0064] FIG. 24 is (a) an SEM and (b) a TEM of commercial sample
11;
[0065] FIG. 25 is (a) an SEM and (b) a TEM of commercial sample
12;
[0066] FIG. 26 is (a) an SEM and (b) a TEM of commercial sample
13;
[0067] FIG. 27 shows SEM images of comparative polymer particles of
two different porosities made using high molecular weight seed
particles; and
[0068] FIG. 28 shows an SEM image of a cross section of a magnetic
polymer particle cast in epoxy, in which the iron oxide is
visualized as bright points.
DETAILED DESCRIPTION
[0069] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of them mean
"including but not limited to", and they are not intended to (and
do not) exclude other moieties, additives, components, integers or
steps. Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0070] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith. All of the features
disclosed in this specification (including any accompanying claims,
abstract and drawings), and/or all of the steps of any method or
process so disclosed, may be combined in any combination, except
combinations where at least some of such features and/or steps are
mutually exclusive. The invention is not restricted to the details
of any foregoing embodiments. The invention extends to any novel
one, or any novel combination, of the features disclosed in this
specification (including any accompanying claims, abstract and
drawings), or to any novel one, or any novel combination, of the
steps of any method or process so disclosed.
[0071] The readers attention is directed to all papers and
documents which are filed concurrently with or previous to this
specification in connection with this application and which are
open to public inspection with this specification, and the contents
of all such papers and documents are incorporated herein by
reference.
[0072] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control.
[0073] The present invention provides novel polymer particles and a
modified Ugelstad process by which the novel particles may be
prepared. It also provides seed particles for use in the modified
Ugelstad process and methods of using the polymer particles which
may be obtained by the modified Ugelstad process.
[0074] The Ugelstad process described herein involves, therefore,
two different particles, namely a seed particle which is subjected
to a swelling and polymerisation process to form a polymer
particle. The terms "seed particle" and "polymer particle" are
therefore used herein as follows:
[0075] "Seed particle" means a particle obtainable by emulsion
polymerisation and used as an intermediate in the modified Ugelstad
process.
[0076] "Polymer particle" refers to a particle which may be made
from the seed particle by suspension polymerisation in the modified
Ugelstad process described herein.
[0077] The mention in this specification of "average" diameters
refers to the z-average diameter, e.g. the z-average diameter
measured by photon correlation spectroscopy. However, across the
entire scope of the invention, there are also hereby disclosed
embodiments in which the average diameters are the mode diameter,
e.g. as measured by CPS disc centrifuge. Across the entire scope of
the invention, there are further hereby disclosed embodiments in
which the average diameters are the mean diameter. However, some
embodiments specify that polymer particles have an average diameter
which falls within at least one of the following two categories:
(i) a specific range of z-average diameters; (ii) a specific range
of mean diameters. Thus, a prescribed class of particles falling
within the disclosure requires the particles to have an average
diameter which falls within at least one of the following two
categories: (i) a z-average diameter of less than 1 .mu.m; (ii) a
mean diameter of less than 1 .mu.m; in such a case, a population of
particles will fall within the prescribed class if it belongs to
category (i) or category (ii) or both categories (i) and (ii).
The Polymer Particle
[0078] The invention provides particles which are polymeric and
monodisperse. The invention includes embodiments in which the
particles are porous and embodiments in which the particles are
solid. Particles of the type comprising a polymer shell over liquid
core containing magnetic particles are not included in the
invention.
[0079] The particles may be in a population of at least 100, e.g.
at least 1000.
[0080] By "monodisperse" is meant that for a plurality of particles
(e.g. at least 100, more preferably at least 1000) the particles
have a coefficient of variation (CV) of their diameters of less
than 20%, for example less than 15%, typically of less than 10% and
optionally of less than 8%, e.g. less than 5%. A particular class
of polymer particles has a CV of less than 5%. CV when referred to
in the claims of this specification is defined as 100 times
(standard deviation) divided by average where "average" is mean
particle diameter and standard deviation is standard deviation in
particle size. The disclosure also includes embodiments where the
"average" is either the z-average or mode particle diameter. In
accordance with usual practice, CV is calculated on the main mode,
i.e. the main peak, thereby excluding minor peaks relating to
aggregates. Thus some particles below or above mode size may be
discounted in the calculation which may for example be based on
about 90% of total particle number (of detectable particles that
is). Such a determination of CV is performable on a CPS disc
centrifuge.
[0081] The invention provides also monodisperse polymer particles
which are of submicron size, i.e. have an average diameter of less
than 1 .mu.m.
[0082] The invention also provides monodisperse polymer particles
having a smooth outer surface.
[0083] More particularly, the invention provides monodisperse
submicron particles having a smooth outer surface. In one
embodiment monodisperse submicron particles have an outer surface
that has a smooth appearance when viewed at a scanning electron
microscope (SEM) magnification of 10,000.
[0084] The polymer particles may be produced by an Ugelstad process
described later in the specification.
[0085] In one embodiment, the invention provides monodisperse
cross-linked polymer particles having the following
characteristics: [0086] a substantially smooth outer surface [0087]
a z-average diameter of less than 1 .mu.m [0088] the particles are
porous [0089] a coefficient of variation (CV) %, when measured by
CPS disk centrifugation analysis, of less than 15%.
[0090] A second embodiment resides in monodisperse cross-linked
polymer particles being a first population of polymer particles and
having the following characteristics: [0091] a substantially smooth
outer surface [0092] a z-average diameter of less than 1 .mu.m
[0093] a coefficient of variation (CV) %, when measured by CPS disk
centrifugation analysis, of less than 15% [0094] a swellability of
not more than the swellability of a second population of polymer
particles, wherein the second population of particles are reference
particles produced by a known Ugelstad process and made of a
comparable polymer to the first population, wherein the amount of
cross-linker monomer used in the suspension polymerisation stage of
the known Ugelstad process is >25% by weight of the total weight
of monomers used in the suspension polymerisation stage, optionally
wherein the first population of polymer particles are polystyrenic
particles and the monomers used in the suspension polymerisation
stage in the preparation of the reference particles were styrene
and, as a crosslinker monomer, divinylbenzene, the divinylbenzene
being in an amount of >25% by weight of the total of styrene
plus divinylbenzene.
[0095] The invention further provides monodisperse cross-linked
polymer particles, having the following characteristics: [0096] a
z-average diameter of less than 1 .mu.m, [0097] a coefficient of
variation (CV) %, when measured by CPS disk centrifugation
analysis, of less than 15% [0098] a specific surface area of from
300 to 700 m.sup.2/g when measured by gas adsorption analysis.
[0099] In a further embodiment, the invention provides monodisperse
cross-linked polymer particles having the following
characteristics: [0100] a substantially smooth outer surface [0101]
an average diameter which falls within at least one of the
following categories (i) a mean diameter of less than 1 .mu.m; (ii)
a z-average diameter of less than 1 .mu.m [0102] the particles are
porous [0103] a coefficient of variation (CV) %, when measured by
CPS disk centrifugation analysis, of less than 15%.
[0104] A further implementation of the invention resides in
monodisperse cross-linked polymer particles being a first
population of polymer particles and having the following
characteristics: [0105] a substantially smooth outer surface [0106]
an average diameter which falls within at least one of the
following categories (i) a mean diameter of less than 1 .mu.m; (ii)
a z-average diameter of less than 1 .mu.m [0107] a coefficient of
variation (CV) %, when measured by CPS disk centrifugation
analysis, of less than 15% [0108] the swellability of a second
population of polymer particles, wherein the second population of
particles are reference particles produced by a known Ugelstad
process and made of a comparable polymer to the first population,
wherein the amount of cross-linker monomer used in the suspension
polymerisation stage of the known Ugelstad process is >25% by
weight of the total weight of monomers used in the suspension
polymerisation stage, optionally wherein the first population of
polymer particles are polystyrenic particles and the monomers used
in the suspension polymerisation stage in the preparation of the
reference particles were styrene and, as a crosslinker monomer,
divinylbenzene, the divinylbenzene being in an amount of >25% by
weight of the total of styrene plus divinylbenzene.
[0109] The invention further provides monodisperse cross-linked
polymer particles, having the following characteristics: [0110] an
average diameter which falls within at least one of the following
categories (i) a mean diameter of less than 1 .mu.m; (ii) a
z-average diameter of less than 1 .mu.m, [0111] a coefficient of
variation (CV) %, when measured by CPS disk centrifugation
analysis, of less than 15% [0112] a specific surface area of from
300 to 700 m.sup.2/g when measured by gas adsorption analysis.
[0113] Reverting now to the polymer particles, this specification
discloses cross-linked polymer particles. It is a characteristic of
cross-linked particles that, when placed in a good solvent for the
polymer, the particles swell instead of dissolving. By way of
example, toluene and THF (tetrahydrofuran) are good solvents for
styrene polymers, whilst THF is also a good solvent for acrylic
polymers. Included in the disclosure are polymer particles
comprising a cross-linked polymer obtainable by a process
calculated to provide at least 2% cross-linking.
[0114] The level of cross-linking in a polymer particle made by the
Ugelstad process can be expressed as the percentage by weight (%
wt) of cross-linker monomer included in the total monomer used in
the suspension polymerisation. Thus, where the monomers used in the
suspension polymerisation are, for example, styrene and
divinylbenzene (DVB) the percentage of DVB (the cross-linker
monomer) is calculated as weight percent based upon the total
weight of DVB plus styrene. Typical levels of cross-linking include
>10% wt cross-linker, for example >15% wt cross-linker, or
>20% wt cross-linker, e.g. >25% wt cross-linker, levels
which, e.g. are suitable for non-porous (i.e. solid) particles. The
level of cross-linking may also be, for instance 20-70% wt
cross-linker, for example 30-60% wt cross-linker, e.g. 40-50% wt
cross-linker, levels which, e.g. are suitable for porous particles.
As stated above, cross-linked particles swell when placed in a good
solvent for the polymer. The amount of swelling, e.g. measured as
an increase in diameter, is related to the level of cross-linking.
Particles with a higher degree of cross-linking will typically
swell less than particles made from a similar polymer, but with a
lower degree of cross-linking. This property can be used to
determine the relative level of cross-linking in a sample of
polymer particles by comparing the sample with a series of
standards of known, different levels of cross-linking. For example,
it may be determined whether a sample of particles have a degree of
cross-linking exceeding 25% by comparing their swellability with
that of reference particles made of a comparable polymer and
prepared using 25% cross-linker as described above. In order to
provide chemically comparable reference particles, the sample of
particles (or, more precisely, a specimen from the sample) is
analysed to determine what class of polymer the sample is made of.
Suitable analytical techniques are mentioned in more detail later
in this specification and may include mass spectrometry, where
pyrolysis mass spectrometry is especially useful to determine
polymer class of cross linked polymers. FTIR and NMR may also be
used in the polymer analysis. The reference particles and the test
particles suitably have substantially similar average diameters,
for example substantially similar z-average diameters, e.g. the
reference particles may have the same average diameter as the test
particles .+-.5%, e.g. .+-.2%.
[0115] In some instances, polystyrenic test particles have their
cross-linking compared with a reference population. The identity of
the test particles as polystyrenic may be known or determined by
analysis. The reference particles are made by an Ugelstad process
in which the monomers used in the suspension polymerisation stage
are styrene and, as a crosslinker, divinylbenzene, the
divinylbenzene being in a known amount, e.g. of 25% by weight of
the total of styrene plus divinylbenzene. The reference particles
and the test particles suitably have substantially similar average
diameters. The test particles and the reference particles are
contacted with a swelling agent, i.e. a suitable solvent, and
allowed to swell. The swelling (increase in diameter) of the test
particles is compared with that of the reference particles and, if
it is found to be less than that of the reference particles, then
the degree of cross-linking is deduced to be higher than in the
reference particles, e.g. higher than 25%. Particles made of any
particular class of polymer will swell different amounts with
different solvents. It is advantageous to select a solvent which
produces a relatively high degree of swelling, and this may be
selected empirically. In the case of polystyrenic particles,
toluene may be used as the swelling agent.
[0116] The invention therefore includes embodiments of the polymer
particles wherein the polymer particles constitute a first
population of particles and have the swellability of a second
population of polymer particles, wherein the second population of
particles are reference particles produced by a known Ugelstad
process and made of a comparable polymer to the first population,
wherein the amount of cross-linker monomer used in the suspension
polymerisation stage of the known Ugelstad process is >25% by
weight of the total weight of monomers used in the suspension
polymerisation stage, optionally wherein the first population of
polymer particles are polystyrenic particles and the monomers used
in the suspension polymerisation stage in the preparation of the
reference particles were styrene and, as a crosslinker monomer,
divinylbenzene, the divinylbenzene being in an amount of >25% by
weight of the total of styrene plus divinylbenzene. The amount of
the cross-linker monomer used in said suspension polymerisation
stage may be 30-60%, optionally 40-50%.
[0117] The particles suitably comprise addition polymer made by
polymerising one or more ethylenically unsaturated monomers. In
particular, the monomers may be vinylic, for example a styrenic
monomer or an acrylic monomer. Styrenic monomers may be mentioned
in particular. Suitable monomers include styrene, methyl
methacrylate, methacrylic acid, hydroxyethyl methacrylate, glycidyl
methacrylate, butylmethacrylate, acrylic acid, ethyleneglycol dim
ethacrylate, trimethylolpropane trimethacrylate, trimethylol
triacrylate, pentaerythritol tetraacrylate and other acrylic or
methacrylic monomers. Cross-linking may be achieved by
incorporating a cross-linker comprising two vinyl groups as a
comonomer, for example divinylbenzene or ethyleneglycol
dimethacrylate. The invention includes the use of a combination of
cross-linkers. As a particular monomer may be mentioned styrene,
for which divinylbenzene is a suitable cross-linker. In some
embodiments, cross-linker may include compounds in which the number
of ethylenic double bonds is greater than two, e.g. three.
[0118] The described Ugelstad processes include polymerisation
reactions which consist of free radical polymerisation of
unsaturated monomers. The disclosure therefore includes polymer
particles wherein the polymer (the matrix polymer) is derived from
radical polymerisation of unsaturated monomers. Cross-linking,
therefore, is obtained by incorporation of cross linkers which
comprise two ethylenic double bonds (as mentioned elsewhere in this
specification, the cross-linker preparation may include molecules
in which the number of ethylenic double bonds is more than two,
e.g. three), and the polymer particles contain the residue of such
cross-linkers incorporated in the polymer network. An alternative
cross-linking method which has been used in the manufacture of
polymer particles is Lewis-acid catalysed Davankov-type
crosslinking which is used in the case of suspension-polymerised
particles. In the Davankov method, particles are made by suspension
polymerisation using vinylbenzyl chloride (VBC) as one of the
monomers. After the particles have been made, they are cross-linked
in a separate reaction by combining the particles with FeCl.sub.3,
which catalyses coupling of a pendant --CH.sub.2Cl group of a
former VBC molecule now incorporated in a polymer chain with a
neighbouring phenyl group, so as to form a methylene bridge between
the two phenyl groups: -Ph-CH.sub.2-Ph-. It will be appreciated
that it is inherent in particles obtainable by the Ugelstad process
that they are free of such -Ph-CH.sub.2-Ph- fragments containing
methylene-bridged benzene rings, and this provides a way of
distinguishing between suspension polymerisation particles made
using Davankov cross-linking and Ugelstad particles. It is
envisaged that the presence or absence of such -Ph-CH.sub.2-Ph-
fragments may be determined by one or more analytical techniques,
for example NMR or mass spectrometry, particularly NMR. The
disclosure therefore includes within its scope monodisperse
submicron particles free of -Ph-CH.sub.2-Ph- fragments. Particles
made by the Ugelstad process may be free of chlorine when subjected
to elemental analysis whereas it is believed that particles made
using the Davankov process will contain residual chlorine in
unreacted --CH.sub.2Cl groups. The disclosure therefore includes
within its scope monodisperse submicron particles free of
chlorine.
[0119] The polymer network produced by Davankov crosslinking of
suspension polymerised particles is therefore different from that
which results from Ugelstad polymerisation. Such structural
differences are reflected in physico-chemical properties, for
example in relation to solvent uptake and swelling properties.
Thus, polymer particles made by suspension polymerisation to form
intermediate gel-type particles followed by Davankov crosslinking
show a much higher degree of swellability than Ugelstad particles,
resulting in large differences of swelling and solvent uptake
between different solvents. Where the polymer particles may be used
in procedures which require switching between solvents, e.g.
between aqueous and organic solvents (e.g. hydrocarbon solvent),
such sharp differences of behaviour are disadvantageous.
[0120] The submicron particles of the invention may have an average
diameter of at least 200 nm, e.g. at least 300 nm, optionally at
least 400 nm, as in the case of particles having a diameter of at
least 450 nm.
[0121] The submicron polymer particles may have an average diameter
of no more than 900 nm, optionally no more than 700 nm, e.g. of no
more than 600 nm, as in the case of particles having a diameter of
no more than 500 nm.
[0122] The invention includes a class of polymer particles having
average diameters of from 400 nm to 800 nm, e.g. 450 nm to 700 nm.
Particular polymer particles have an average diameter of from 450
nm to 650 nm, e.g. 450 nm to 550 nm. In embodiments, the polymer
particles may have an average diameter of from 200 nm, e.g. 300 nm
and optionally 400 nm, to 450 nm.
[0123] The size and size distribution of seed particles may be
determined as described below under the heading "analytical
methods".
[0124] One class of polymer particles is porous. Another class of
polymer particles is non-porous. The porous particles and,
independently, the non-porous particles may have average diameters
as just indicated.
[0125] The disclosure includes porous polymer particles having a
specific surface area of from 100 to 700 m.sup.2/g, for example
from 300 to 600 m.sup.2/g, or from 400 to 600 m.sup.2/g, e.g. from
450 to 550 m.sup.2/g. The specific surface area values are
determined by gas adsorption analysis.
[0126] The disclosure includes porous polymer particles having a
ratio of specific surface area, as measured by gas adsorption
analysis, to theoretical specific surface area for a compact
particle, of at least 10:1, for example of from 10:1 to 150:1, e.g.
from 10:1 to 120:1 as in the case of 10:1 to 110:1. The ratio may
be at least 20:1 and is optionally at least 50:1 e.g. at least
80:1. Thus, the ratio may be from 20:1 or 50:1 to 150:1, whilst in
other embodiments it is from 20:1 to 120:1 or alternatively from
50:1 to 120:1. In some embodiments, the ratio is from 80:1 to
150:1, e.g. from 80:1 to 120:1, as in the case of 80:1 to
110:1.
[0127] The disclosure includes magnetic polymer particles having a
specific surface area of greater than 30 m.sup.2/g, for example
greater than 35 or 40 m.sup.2/g. The specific surface area may for
example be from greater than 30 m.sup.2/g (e.g. greater than 40
m.sup.2/g) to 100 m.sup.2/g, for example to 90 m.sup.2/g; in some
embodiments the specific surface area does not exceed 80 m.sup.2/g
and in particular embodiments it does not exceed 70 m.sup.2/g.
Particles having such properties are obtainable by forming porous
particles by the novel methods described herein and then
incorporating magnetic material in the pores and coating the
particles.
[0128] The disclosure includes magnetic polymer particles having a
ratio of specific surface area, as measured by gas adsorption
analysis, to theoretical specific surface area for a compact
particle, of at least 2:1, for example of from 2:1 to 20:1, e.g.
from 2:1 to 18:1 as in the case of 2:1 to 16:1. The ratio may be at
least 3:1 and is optionally at least 4:1. Thus, the ratio may be
from 3:1 or 4:1 to 20:1, whilst in other embodiments it is from 3:1
to 16:1 or alternatively from 4:1 to 16:1. In some embodiments, the
ratio is from 2:1 to 14:1, e.g. from 3:1 to 14:1.
[0129] In the case of non-porous polymer particles, the disclosure
includes particles having a specific surface area of less than 20
m.sup.2/g. In one embodiment the non-porous particles have a
specific surface area of from 2 to 20 m.sup.2/g, particularly of
from 5 to 20 m.sup.2/g, e.g. from 6 to 20 m.sup.2/g. In another
embodiment, the specific surface area is from 2 to 10, e.g. 2 to 8
m.sup.2/g. By way of example, particles having a density of 1.05
g/ml and a diameter of from 900 to 300 nm would have a specific
surface area of from about 6 to about 20 m.sup.2/g. Exemplary
specific surface areas are as follows:
[0130] Diameter--0.3 .mu.m, density 2.5 g/mL-->A.sub.s=8.0
m.sup.2/g
[0131] Diameter--0.9 .mu.m, density 2.5 g/mL-->A.sub.s=2.7
m.sup.2/g
[0132] Diameter--0.3 .mu.m, density 1.8 g/mL-->A.sub.s=11.1
m.sup.2/g
[0133] Diameter--0.9 .mu.m, density 1.8 g/mL-->A.sub.s=3.7
m.sup.2/g
[0134] Diameter--0.3 .mu.m, density 1.05 g/mL-->A.sub.s=19.0
m.sup.2/g
[0135] Diameter--0.9 .mu.m, density 1.05 g/mL-->A.sub.s=5.7
m.sup.2/g.
[0136] For porous coated magnetic particles the specific surface
area will normally be at least twice the specific surface area of
the corresponding compact particle having the same mean particle
size and density. A typical density for coated magnetic beads is
1.8 g/mL The magnetic beads can have lower density (less magnetic
material incorporated) or higher density. In embodiments, a maximum
density for a coated magnetic bead would be 2.5 g/mL.
[0137] The measurement of specific surface areas is described later
in the specification under the heading "analytical methods".
[0138] Porous polymer particles may comprise a magnetic material in
the pores, for example one or more magnetic materials. By
incorporating the magnetic material in the pores, it is possible to
retain the smooth appearance of smooth porous submicron particles
of the disclosure. The invention is not limited as to the identity
of the magnetic material, e.g. the magnetic material may comprise
at least one of a paramagnetic, superparamagnetic, ferromagnetic or
ferrimagnetic material. One class of polymer particles comprises a
paramagnetic material. Another class of polymer particles comprises
a superparamagnetic material. A further class of polymer particles
comprises ferromagnetic material, ferrimagnetic material or both.
At this point, it may be helpful to refresh the reader's knowledge
of these terms:
[0139] Magnetic: responds to a magnetic field.
[0140] Paramagnetic: the magnetic properties are switched off when
the magnetic field is removed.
[0141] Superparamagnetic: the switching off of the magnetic
properties with removal of the magnetic field is very
rapid/instant.
[0142] Ferromagnetic: all of its magnetic atoms within each domain
add a positive contribution to the net magnetization. Retains
magnetic properties after an external magnetic field is removed.
Above the Curie temperature becomes a paramagnetic material.
[0143] Ferrimagnetic: some magnetic atoms within each domain are
opposed, but overall exhibits net magnetization. Retains magnetic
properties after an external magnetic field is removed. Above the
Curie temperature becomes a paramagnetic material.
[0144] In one class of embodiments, therefore, the polymer
particles described herein comprise superparamagnetic material,
particularly superparamagnetic crystals. The superparamagnetic
crystals of the polymer particles may be of any material capable of
being deposited in superparamagnetic crystalline form on the
polymer particles or in the pores thereof, should the particle be
porous The magnetic material may comprise, or be, an iron oxide,
for example a ferrite such as, e.g. magnetite or maghemite, or a
combination thereof. A portion of the iron in the iron oxide, e.g.
magnetite or maghemite, may be substituted by (i.e. replaced with)
Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti or Sn or a combination
thereof. In particular, magnetite or maghemite may have a portion
of their iron substituted by Mn. As mentioned, some particles
contain a combination of magnetite and maghemite; in this case,
either the magnetite or the maghemite, or both, may have such
partial substitution of their iron content. Additionally or
alternatively, particles of the disclosure may include iron-based
metal nanoparticles and FeNi alloy nanoparticles, in either case to
increase the saturation magnetization of the particles. Where a
portion of the iron is substituted by one or more other elements
the total amount substituted can be up to 5 mol %, for example in
the range of 0.1 to 5 mol %, for example 0.5 to 4 mol %, e.g. 1 to
3 mol %.
[0145] In another class of embodiments, the polymer particles are
ferrimagnetic and in particular may comprise, or be, ferrimagnetic
iron oxide crystals. Accordingly, the disclosure includes polymer
particles as described herein which comprise magnetic particles
selected from ferrimagnetic iron oxide crystals, superparamagnetic
iron oxide crystals and combinations thereof.
[0146] The magnetic particles of a polymer particle may comprise,
or be, iron oxide crystals.
[0147] The total quantity of magnetic material present is generally
more than 20%, preferably more than 25%, desirably more than or
equal to 30%, e.g. up to 85% wt or at least 50 wt %, e.g. 30 to 80
wt %. The percentage is a weight percentage calculated on the
weight of magnetic material (e.g. metal oxides) based upon the
overall dry weight of the particles. Where the magnetic material
consists of superparamagnetic material, therefore, the total
quantity of superparamagnetic material present is generally more
than 20%, preferably more than 25%, desirably more than or equal to
30%, e.g. up to 85% wt or at least 50 wt %, e.g. 30 to 80 wt %, the
percentages each being a weight percentage calculated on the weight
of magnetic material (e.g. metal oxides) based upon the overall dry
weight of the particles.
[0148] The outer surfaces of the polymer particles may be coated.
As exemplary coatings may be mentioned those formed by reacting
surface-functionalized particles with an epoxide monomer or with a
polyisocyanate, e.g. diisocyanate, and a diol, as described in WO
2004/053490, WO 2005/015216 or WO 2006/075185, the contents of all
of which are incorporated herein by reference in their entirety.
Other exemplary coatings include metal coatings, e.g. gold plating
or silver, copper, zinc or tin coatings, as described in WO
00/24005 (incorporated herein by reference in its entirety).
[0149] The incorporation of magnetic particles in the particle
pores and the coating of particles do not substantially change
external morphology, i.e. smooth porous submicron particles remain
visually smooth when viewed by SEM at a magnification of 10,000. It
is a characteristic of porous polymer particles obtainable by the
Ugelstad process and having magnetic material incorporated as
described herein (see under the heading below "Preparation of
Particles" for a description of the methods of incorporating
magnetic material) that the magnetic material is distributed
throughout the particle in a relatively homogenous way, as shown in
FIG. 29. The Figure shows that the iron oxide particles are
dispersed throughout the polymer particles without clumping and
that, whilst the density of iron oxide particles appears to reduce
towards the centre of the particle, there is no variation in
density in a circumferential direction. In view of the absence of
clumping and the wide dispersal of the particles, the distribution
of the magnetic material may for practical purposes be regarded as
homogeneous.
[0150] For the magnetised and coated, originally porous, particles,
there are two distinct properties that generally reflect the porous
nature of the polymer particle. [0151] 1) the magnetic material,
e.g. iron oxide, is distributed evenly through the polymer bead (in
pores) as discussed in the previous paragraph [0152] 2) magnetised
and coated, originally porous, polymer particles will have a
specific surface area which is slightly larger than the specific
surface area of a corresponding compact polymer particle.
[0153] A corresponding compact polymer particle (same diameter and
density) will have a theoretical specific surface area A.sub.s:
A = 6 D m .rho. for m = 1 g A s = 6 D .rho. , ##EQU00001## [0154]
where [0155] D=particle diameter (.mu.m) [0156] .rho.=density
(g/mL) [0157] m=sample weight (g) [0158] A=surface area (m.sup.2)
[0159] A.sub.s=specific surface area (m.sup.2/g).
[0160] The density of the coated polymer particle can be determined
by a helium pycnometer.
[0161] Typical values for the specific surface area of coated and
functionalised magnetic particles made using the Ugelstad method
are:
[0162] 1 .mu.m particles: 6-30 m.sup.2/g
[0163] 0.5 .mu.m particles of the invention: 10-70 m.sup.2/g.
The invention therefore includes in some implementations magnetic
particles having a diameter of less than 1 .mu.m and a specific
surface area of greater than 30 m.sup.2/g, for example greater than
35 or 40 m.sup.2/g. The specific surface area may for example be
from greater than 30 m.sup.2/g (e.g. greater than 40 m.sup.2/g) to
100 m.sup.2/g, for example to 90 m.sup.2/g; in some embodiments the
specific surface area does not exceed 80 m.sup.2/g and in
particular embodiments it does not exceed 70 m.sup.2/g.
[0164] Additionally or alternatively to coating, the surfaces of
the polymer particles may be provided with a functional group or a
combination of functional groups, for example selected from a
carboxylic acid, amine, tosyl, epoxy or hydroxy group. Such
functionalisation enables further reaction of the particles, e.g.
enables conjugation of substances to the particles.
[0165] If desired, therefore, the surfaces of the coated magnetic
polymer particles may be further conjugated with a desired
substance, e.g. by coupling a drug molecule, a reporter label (e.g.
a chromophore, fluorophore, enzyme or radiolabel), or a ligand
(e.g. an antibody or antibody fragment, a metal ion complexing
agent, a member of a specific binding partner pair (e.g. biotin or
streptavidin), an oligopeptide, an oligonucleotide, or an
oligosaccharide.
[0166] Such coupling may be direct or indirect (and so may or may
not involve the use of a coupling agent to form a linkage between
the particle and the substance being coupled to it) and may be
biodegradable or non-biodegradable. Biodegradable couplings may be
desired if the polymer particles are to be used for the targeted
release of an active compound. Where such derivatisation is
desired, functional groups must be provided on the particles for
the attachment of conjugated substances; for example, where the
particle is coated, pendent groups of the coating may be
manipulated to provide appropriate functionality (for example
carboxy, epoxy, hydroxy, amino, tosyl etc. functionalities).
[0167] The functionalised coated magnetic particle may be bound to
an affinity ligand the nature of which will be selected based on
its affinity for a particular analyte whose presence or absence in
a sample is to be ascertained. The affinity molecule may therefore
comprise any molecule capable of being linked to a magnetic probe
which is also capable of specific recognition of a particular
analyte. Affinity ligands therefore include monoclonal antibodies,
polyclonal antibodies, antibody fragments, nucleic acids,
oligonucleotides, proteins, oligopeptides, polysaccharides, sugars,
peptides, peptide encoding nucleic acid molecules, antigens, drugs
and other ligands. Examples of suitable affinity ligands are
available in the published literature and are well known. The use
of further binding partners, secondary affinity ligands and linking
groups which is routine in the art will not be discussed further
herein although it will be appreciated that the use of such species
with the particles of the invention is possible if desired.
[0168] It will be appreciated from the aforegoing that the
particles of the disclosure, which may be in a population of at
least 100, e.g. of at least 1,000, include particles characterised
by a z-average diameter of less than 1 .mu.m and monodispersity,
which is conveniently defined as a coefficient of variation (CV) %,
when measured by CPS disk centrifugation analysis, of less than 15%
and optionally of less than 12%, e.g. of less than 10% as in the
case of less than 5%. The particles may be characterized in terms
of one of more additional properties described herein which,
individually or in various combinations, distinguish the particles
from those in the prior art: [0169] (i) a z-average diameter
falling within a sub-class mentioned previously, e.g. (a) of no
more than: 900 nm, 700 nm, 600 nm or 500 nm and/or (b) of at least
200 nm and/or (c) of from 400-800 nm, 450-700 nm, 450-650 nm,
450-550 nm, 200-450 nm, or 300-450 nm; [0170] (ii) a substantially
smooth outer surface when viewed by SEM at a magnification of
10,000, e.g. a surface morphology substantially as it appears in
FIGS. 4-6, 10C and 10D; [0171] (iii) a characteristic, e.g.
swellability, indicating a degree of cross-linking corresponding to
that of a particle having a comparable matrix polymer and made by
the Ugelstad process wherein the level of cross-linking greater
than 25% (e.g. at least 30% such as, for example, 30-60% or 40-50%)
expressed as the percentage by weight of cross-linker monomer
included in the total monomer (monomer and cross-linker monomer)
used in the suspension polymerisation part of the Ugelstad process,
in particular where the monomers used in the suspension
polymerisation are styrene and, as cross-linker, divinylbenzene;
[0172] (iv) the polymer of which the body of the particle is made,
i.e. the matrix polymer, is selected from polymers and copolymers
of monomers selected from acrylic monomers (e.g. methacrylate) and
styrenic monomers (e.g. styrene); [0173] (v) the particle is
porous, compact or magnetic (a magnetic particle is a former porous
particle now containing magnetic material in its pores and usually
coated).
[0174] In the following embodiments, the particles comprise the
following features:
TABLE-US-00001 Embodiment Features A (i) B (ii) C (iii) D (iv) E
(i), (ii) F (iii), (ii) G (iv), (ii) H (i), (iii) I (i), (iv) J
(iii), (iv) K (i), (ii), (iii) L (i), (ii), (iv) M (i), (iii), (iv)
N (ii), (iii), (iv) O (i), (ii), (iii), (iv)
[0175] Porous particles may have one or more of the following
characteristics: [0176] (vi) a specific surface area determine by
gas adsorption analysis of from 100 to 700 m.sup.2/g, 300 to 600
m.sup.2/g, 400 to 600 m.sup.2/g, or 450 to 550 m.sup.2/g; [0177]
(vii) a ratio of specific surface area, as measured by gas
adsorption analysis, to theoretical specific surface area for a
compact particle, of at least 10:1 (for example 10:1 to 150:1, 10:1
to 120:1, or 10:1 to 110:1), of at least 20:1 (for example 20:1 to
150:1, 20:1 to 120:1, or 20:1 to 110:1), of at least 50:1 (for
example 50:1 to 150:1, 50:1 to 120:1, or 50:1 to 110:1), or of at
least 80:1 (for example 80:1 to 150:1, 80:1 to 120:1, or 80:1 to
110:1).
[0178] In the following embodiments, the porous particles comprise
the following features, where e.g. "A+(vi), (vii)" designates a
combination of the features of embodiment A above with features
(vi) and (vii):
TABLE-US-00002 Embodiment Features P (vi) Q (vii) R (vi), (vii) S A
+ (vi) T A + (vii) U A + (vi), (vii) V B + (vi) W B + (vii) X B +
(vi), (vii) Y C + (vi) Z C + (vii) AA C + (vi), (vii) AB D + (vi)
AC D + (vii) AD D + (vi), (vii) AE E + (vi) AF E + (vii) AG E +
(vi), (vii) AH F + (vi) AI F + (vii) AJ F + (vi), (vii) AK G + (vi)
AL G + (vii) AM G + (vi), (vii) AN H + (vi) AM H + (vii) AO H +
(vi), (vii) AP I + (vi) AQ I + (vii) AR I + (vi), (vii) AS J + (vi)
AT J + (vii) AU J + (vi), (vii) AV K + (vi) AW K + (vii) AX K +
(vi), (vii) AY L + (vi) AZ L + (vii) BA L + (vi), (vii) BB M + (vi)
BC M + (vii) BD M + (vi), (vii) BE N + (vi) BF N + (vii) BG N +
(vi), (vii) BH O + (vi) BI O + (vii) BJ O + (vi), (vii)
[0179] Magnetic particles may have one or more of the following
characteristics: [0180] (viii) a specific surface area determine by
gas adsorption analysis of greater than 30 m.sup.2/g, for example
greater than 35 m.sup.2/g or 40 m.sup.2/g (e.g. from more than 30
m.sup.2/g to 100 m.sup.2/g, from 40 m.sup.2/g to 100 m.sup.2/g,
from more than 30 m.sup.2/g to 90 m.sup.2/g, from 40 m.sup.2/g to
90 m.sup.2/g, from more than 30 m.sup.2/g to 80 m.sup.2/g, from 40
m.sup.2/g to 80 m.sup.2/g, from more than 30 m.sup.2/g to 70
m.sup.2/g, from 40 m.sup.2/g to 70 m.sup.2/g); [0181] (ix) a ratio
of specific surface area, as measured by gas adsorption analysis,
to theoretical specific surface area for a compact particle, of at
least 2:1, (e.g. from 2:1 to 20:1, 2:1 to 18:1, 2:1 to 14:1),
optionally of at least 3:1 (e.g. from 3:1 to 20:1, 3:1 to 18:1, 3:1
to 14:1) and is optionally at least 4:1 (e.g. from 4:1 to 20:1, 4:1
to 18:1, 4:1 to 14:1).
[0182] In the following embodiments, the magnetic particles
comprise the following features, where e.g. "A+(vi), (vii)"
designates a combination of the features of embodiment A above with
features (vi) and (vii):
TABLE-US-00003 Embodiment Features BK (viii) BL (ix) BM (viii),
(ix) BN A + (viii) BO A + (ix) BQ A + (viii), (ix) BR B + (viii) BS
B + (ix) BT B + (viii), (ix) BU C + (viii) BW C + (ix) BX C +
(viii), (ix) BY D + (viii) BZ D + (ix) CA D + (viii), (ix) CB E +
(viii) CD E + (ix) CE E + (viii), (ix) CF F + (viii) CG F + (ix) CH
F + (viii), (ix) CI G + (viii) CJ G + (ix) CK G + (viii), (ix) CL H
+ (viii) CM H + (ix) CN H + (viii), (ix) CO I + (viii) CP I + (ix)
CQ I + (viii), (ix) CR J + (viii) CS J + (ix) CT J + (viii), (ix)
CU K + (viii) CV K + (ix) CW K + (viii), (ix) CX L + (viii) CY L +
(ix) CZ L + (viii), (ix) DA M + (viii) DB M + (ix) DC M + (viii),
(ix) DD N + (viii) DE N + (ix) DF N + (viii), (ix) DG O + (viii) DI
O + (ix) DJ O + (viii), (ix)
[0183] For all embodiments, any one or more compatible features
mentioned earlier in this specification may be included, for
example attachment of functional groups, silica coating, absence of
methylene bridged benzene rings etc.
Seed Particles
[0184] The polymer particles may be prepared by the Ugelstad
process, starting with specific seed particles. The polymer seed
particles are monodisperse and have an average diameter of from 20
nm to 200 nm, and the polymer, e.g. polystyrene, has a mean weight
average molecular weight of greater than 1,000 but less than
70,000, when measured by gel permeation chromatography.
Additionally or alternatively to the specified molecular weight
range, the polymer may comprise about 10 to 700 monomer units. In
embodiments, the average diameter of the seed particles is from 50
nm to less than 200 nm, e.g. 50 nm to 190 nm.
[0185] The mean weight average molecular weight of the seed
particle polymer may be less than 40,000, optionally less than
30,000, further optionally less than 20,000, e.g. less than 15,000.
The mean weight average molecular weight may be more than 2,000,
optionally more than 4,000, further optionally more than 6,000, as
in the case of more than 8,000, e.g. more than 10,000. For example,
the mean weight average molecular weight may be from 6,000 to
70,000, e.g. from 6,000 to 40,000, for example from 8,000 to 70,000
or from 8,000 to 40,000.
[0186] In particular embodiments, the mean weight average molecular
weight of the polymer of the seed particles is from 8,000 to
20,000.
[0187] The polymer may comprise fewer than 400 monomer units,
optionally fewer than 300 monomer units, further optionally fewer
than 200 monomer units, e.g. fewer than 150 monomer units. The
polymer may comprise more than 20 monomer units, optionally more
than 40 monomer units, further optionally more than 60 monomer
units, as in the case of more than 80 monomer units, e.g. more than
100 monomer units.
[0188] In particular embodiments, the polymer of the seed particle
has from 80 to 200 monomer units.
[0189] The CV of the seed particle diameters, when measured by CPS
disc centrifugation analysis, may be less than 10%, optionally less
than 5%, e.g. less than 2% in some cases.
[0190] The seed particles suitably comprise or consist of addition
polymer made by polymerising one or more ethylenically unsaturated
monomers. In particular, the monomers may be vinylic, for example a
styrenic monomer or an acrylic monomer. Styrenic monomers may be
mentioned in particular. Suitable monomers include styrene, methyl
methacrylate, methacrylic acid, hydroxyethyl methacrylate, glycidyl
methacrylate, butylmethacrylate and acrylic acid and other acrylic
or methacrylic monomers.
[0191] The seed particles in particular may consist of
polystyrene.
Preparation of Particles
[0192] The seed particles may be made by emulsion polymerisation,
following in general the procedures described in Example 9 of WO
00/61647 (incorporated herein by reference in its entirety) but
with modifications to control the molecular weight of the polymer
of the seed particles and the diameter of the seed particles. The
preparation may be carried out under substantially oxygen-free
conditions as described in WO 00/61647, but alternatively is
carried out in the presence of oxygen.
[0193] The seed particles may therefore be made by an emulsion
polymerisation process comprising:
[0194] forming an aqueous dispersion comprising an ethylenically
unsaturated monomer and a water soluble polymerisation initiator;
and
[0195] agitating, e.g. mixing, until the commencement of particle
nucleation; characterised in that:
[0196] the aqueous dispersion comprises a surfactant and in that a
chain transfer agent is added after the commencement of particle
nucleation, such that the polymerisation forms monodisperse seed
particles having an average diameter of from 50 nm to 200 nm and,
when measured by gel permeation chromatography, the polymer has a
mean weight average molecular weight of more than 1,000 and less
than 70,000.
[0197] The ethylenically unsaturated monomer may be vinylic, for
example a styrenic monomer or an acrylic monomer. Styrenic monomers
may be mentioned in particular. Suitable monomers include styrene,
methyl methacrylate, methacrylic acid, hydroxyethyl methacrylate,
glycidyl methacrylate, butylmethacrylate and acrylic acid and other
acrylic or methacrylic monomers.
[0198] The polymerisation initiator may be any water soluble
initiator. A suitable initiator is a persulfate (also known as
peroxodisulfate), e.g. potassium or ammonium persulfate.
[0199] The surfactant may be ionic or non-ionic and is in
particular ionic, for example sodium dodecyl sulfate (SDS).
[0200] The chain transfer agent may be a haloalkane, for example
bromotrichloromethane, or an alkyl mercaptan, e.g. butyl mercaptan,
isooctyl 3-mercaptopropionate or octylmercaptan
(1-octanethiol).
[0201] Other optional components may be included as desired in the
reaction mixture, in particular a buffer. Borax, i.e. sodium
borate, sodium tetraborate, disodium tetraborate, and/or hydrates
thereof e.g. tetraborate decahydrate, is a suitable buffer, or
another buffer may be used.
[0202] Where it is desired to reduce or substantially prevent
exposure to oxygen, the polymerisation reaction may be carried out
under an oxygen-free atmosphere (e.g. a noble gas). The oxygen
content of the aqueous phase may be reduced by boiling the water
before use or water and/or other liquid reagents may be purged with
nitrogen before use. The oxygen content of the liquid reagents may
also be reduced by purging with an oxygen-free atmosphere
comprising another inert gas, e.g. argon. One or both of these two
measures (use of an oxygen free atmosphere and de-oxygenation of
water and optionally other liquids) may be performed.
[0203] In embodiments, the preparation comprises combining: a
monomer, for example styrene; a buffer, for example borax; a
surfactant, particularly SDS; a water soluble polymerisation
initiator, for example a persulfate; and water, to form an
emulsion, and agitating, e.g. mixing, until particle nucleation
commences. After particle nucleation has started, a chain transfer
agent is added, for example bromotrichloromethane or an alkyl
mercaptan.
[0204] Where desired, any one or more of the reagent classes
(monomer, buffer, surfactant, polymerisation initiator, chain
transfer agent) may comprise a combination of compounds.
[0205] In embodiments, the surfactant is below its critical micelle
concentration (CMC), for example below its CMC in aqueous solution.
The CMC of a surfactant is a function of temperature. For example
when SDS is used as the surfactant, its CMC at 25.degree. C. is 2.3
g/L of water. Thus in embodiments the concentration of SDS is less
than 2.3 g/L of water, optionally less than 2 g/L of water.
[0206] The disclosure includes methods in which the surfactant is
present in a concentration of not more than 2.5 g/L, optionally not
more than 2 g/L, further optionally not more than 1.7 g/L, still
further optionally not more than 1.5 g/L. Also included are methods
in which the surfactant is present in a concentration of at least
0.5 g/L, optionally at least 0.8 g/L, further optionally at least 1
g/L, still further optionally at least 1.2 g/L, even further
optionally at least 1.5 g/L. For example, the surfactant may be
present in an amount of from 0.5 g/L, optionally at least 0.8 g/L,
to 2.5 g/L. In some methods, the surfactant is present in an amount
of from 0.8 g/L, optionally at least 1 g/L, to 2.3 g/L, for example
as in the case where the surfactant is present in an amount of from
0.8 g/L, optionally at least 1 g/L, to 2 g/L, e.g. wherein the
concentration does not exceed 1.7 g/L.
[0207] The amount of the surfactant controls the particle size and
is selected to give the desired average diameter. In this regard,
it has been found that particle size decreases as the concentration
of surfactant is increased. For example, where styrene is selected
as the monomer and SDS as the surfactant, an increase in the SDS
concentration from 1 g/L to 2 g/L was found in one experiment to
reduce average particle size from about 180 nm to about 55 nm. The
concentration of a surfactant that will provide particles with the
desired average particle diameter can be determined
empirically.
[0208] The addition of a chain transfer agent reduces the molecular
weight of the polymer of the seed particles by reacting with the
free radical of a growing polymer chain to terminate the chain and
transfer the lone electron to a radical species derived from the
chain transfer agent. The total amount of chain transfer agent
added can be in the range 1 mol per 10 mol of monomer to 1 mol per
300 mol of monomer, for example 1 mol per 20 mol of monomer to 1
mol per 100 mol monomer, e.g. approximately 1 mol chain transfer
agent per 30 mol of monomer. The time of addition of the chain
transfer agent is important to obtain monodisperse seed particles:
the chain transfer agent should be added shortly after the particle
nucleation step and may be added over an extended time period or
all the agent may be added at substantially the same time. The
particle nucleation step can be detected by the presence of visible
particle nucleation, e.g. by the solution becoming cloudy. If the
chain transfer agent is added before particle nucleation,
polydisperse seed particles may be formed. If all the chain
transfer agent is added at the same time (e.g. at a single
timepoint), a two peak molecular weight distribution plot is
obtained (see FIG. 8). A suitable time to start adding the chain
transfer agent is about 5 to 15 minutes after particle nucleation,
e.g. about 10 minutes after particle nucleation. If the chain
transfer agent is added over an extended time period, it is
typically added for 20 minutes to 4 hours, for example for 30
minutes to 1.5 hours, e.g. for about 40 minutes. The rate of
addition may be constant or variable. Where the chain transfer
agent is added over a suitably extended period, a single peak may
be obtained in the molecular weight distribution plot.
[0209] The monodisperse seed particles can then be directly
subjected to an Ugelstad process, for example as outlined in FIG.
2. FIG. 2 is conveniently divided into 3 steps, activation of the
seed particles (step A), swelling of the activated seed particles
(step B) and polymerisation of the monomer in the swollen seed
particles (step C). Step A involves contacting an aqueous
dispersion of monodisperse polymeric seed particles (1) with an
emulsion comprising finely divided droplets of an organic compound
with low water solubility (2), e.g. water solubility at 25.degree.
C. of less than 10.sup.-2 g/L and/or water solubility of less than
1/1000.sup.th that of a monomer (6) used in a subsequent step of
the procedure. The mixture comprising the aqueous dispersion and
emulsion can also comprise an organic solvent, which it is believed
assists in transporting the organic compound into the seed
particles. The organic compound diffuses into the seed particles
over a period of time (for instance 12 to 36 hours, e.g., 24 hours,
during which the mixture can be agitated, e.g. by stirring),
forming activated seed particles (3). Where present the organic
solvent is then removed, e.g. by dilution. The activated seed
particles (3) in aqueous dispersion are in step B mixed with an
aqueous vehicle containing a monomer (4), e.g. are mixed with an
emulsion comprising droplets of a monomer (4). It will be
appreciated that the mixing of step B can provide removal by
dilution of the organic solvent in the Ugelstad method, for example
the activated seed particles are typical contacted with an excess
of emulsion to dilute the organic solvent, e.g. the weight of the
emulsion may be at least about 5 times greater than that of the
suspension containing the seed particles, e.g. about 10 times
greater (for example 9 times greater). The monomer diffuses into
the activated seed particles, providing swollen particles (5). The
swollen particles (5) comprise a mixture of at least the monomer
(6), the organic compound (7) and polymer from the seed particle
(8). The swollen particle may also include other components, for
instance one or more porogens or cross linkers, which can enter the
particles if included in the mixture during step B. In step C
polymerisation of the monomer is initiated, creating polymer (9)
from the monomer inside the swollen particles (6).
[0210] Accordingly, the invention includes a method of making
submicron monodisperse particles having a substantially smooth
outer surface and an average diameter of less than 1 .mu.m, the
method comprising performing the Ugelstad process using seed
particles having an average diameter of from 50 nm to 200 nm and
made of polymer having a mean weight average molecular weight of
more than 1,000 and less than 70,000, when measured by gel
permeation chromatography.
[0211] Suitably, the Ugelstad process comprises: [0212] (i) forming
an aqueous dispersion comprising [0213] the monodisperse seed
particles, [0214] finely divided droplets comprising an organic
compound of molecular weight below 5,000 and water solubility at
25.degree. C. of less than 10.sup.-2 g/L, and [0215] an organic
solvent in which the organic compound is soluble, the organic
solvent being optional when the polymer forming the seed particles
has an average molecular weight which corresponds to up to 50
monomer units; [0216] (ii) allowing the organic compound to diffuse
into the monodisperse seed particles, causing the seed particles to
become activated; [0217] (iii) removing the organic solvent, where
present, from inside the seed particles, and contacting the
activated seed particles with an aqueous vehicle containing (a) a
monomer having a solubility in water at least 10 times that of the
organic compound and (b) a cross-linker; [0218] (iv) allowing the
monomer and the cross-linker to diffuse into the activated seed
particles to form an aqueous dispersion of swollen seed particles;
and [0219] (v) initiating polymerisation of the monomer and the
cross-linker in the swollen seed particles.
[0220] The monomer used in step (iii) is usually much more soluble
in water than is the organic compound. For example, it may be at
least ten times more soluble in terms of weight per unit volume of
water.
[0221] The cross-linker used in step (iii) is usually much more
soluble in water than is the organic compound. For example, it may
be at least ten times more soluble in terms of weight per unit
volume of water.
[0222] As mentioned, removal of the organic solvent normally
involves dilution of the organic solvent by the aqueous vehicle and
step (iii) may be restated as "contacting the activated seed
particles with an aqueous vehicle containing (a) a monomer having a
solubility in water at least 10 times that of the organic compound
and (b) a cross-linker".
[0223] It is possible to perform the Ugelstad process with more
than one swelling and polymerisation stage. In these embodiments,
the Ugelstad process comprises a first and a final pass, optionally
at least one intermediate pass between the first and final pass, of
the following steps (i) to (vi): [0224] (i) forming an aqueous
dispersion comprising [0225] the monodisperse seed particles (first
pass) or intermediate particles (final pass and optional
intermediate pass), [0226] finely divided droplets comprising an
organic compound of molecular weight below 5,000 and water
solubility at 25.degree. C. of less than 10.sup.-2 g/L, and [0227]
an organic solvent in which the organic compound is soluble, the
organic solvent being optional when the polymer forming the seed
particles has an average molecular weight which corresponds to up
to 50 monomer units; [0228] (ii) allowing the organic compound to
diffuse into the monodisperse seed particles, causing the seed
particles to become activated; [0229] (iii) removing the organic
solvent, where present, from inside the seed particles, and
contacting the activated seed particles or intermediate particles
with an aqueous vehicle containing a monomer having a solubility in
water at least 10 times that of the organic compound and, in the
final pass only, a cross-linker; [0230] (iv) allowing the monomer
and, in the final pass only, the cross-linker to diffuse into the
activated seed particles or intermediate particles to form an
aqueous dispersion of swollen seed particles or intermediate
particles; and [0231] (vi) initiating polymerisation of the monomer
and, in the final pass only, the cross-linker in the swollen seed
particles.
[0232] Steps (i) to (v) are performed once for each pass, with the
particles formed at step (v) representing intermediate particles
suitable for use in step (i) of the subsequent pass, for all but
the final pass. Ugelstad processes with more than one swelling and
polymerisation stage typically involve two or three swelling and
polymerisation stages, i.e. two or three passes of the above
procedures.
[0233] Advantageously, the organic compound is a polymerisation
initiator and desirably a heat-activated polymerisation initiator.
Optionally the heat-activated polymerisation initiator is an
organic peroxide, for example dioctanoylperoxide.
[0234] Where porous particles are desired, a porogen should be
incorporated in the swollen seed particles, preferably in at least
the final swelling and polymerisation stage ((iii)-(v)). As
porogens can be used organic substances which are not polymerised
in the polymerisation stage and which can be removed from the
particles after polymerisation thereby producing porous particles.
Porogens can also be used as blowing agents--particles impregnated
with such materials, on heating may expand as the porogen
vaporizes. Examples of suitable porogens include organic acids,
alcohols, esters, aromatic solvents, optionally substituted
aliphatic hydrocarbons having up to 12 carbons, e.g. toluene,
cyclohexanol, butyl acetate, propane, pentane, cyclopentane,
cyclobutane, heptane, methyl chloride, ethyl chloride,
dichlorodifluoromethane, etc. As a particular example of a porogen
may be mentioned toluene. A porogen may comprise a combination of
compounds.
[0235] Step (iii) may therefore include contacting the seed
particles with a porogen. In particular, the seed particles may be
combined with an emulsion comprising water, monomer, cross-linker,
and a porogen. The emulsion typical contains also a surfactant, for
example SDS or another ionic surfactant. The resulting reaction
mixture is maintained at a moderate temperature (e.g. no more than
30.degree. C., typically at for example 30.degree. C.), for example
for a period of from 1 to 30 hours (typically from 10 to 24 hours),
and polymerisation is then initiated. Where the organic compound
used to swell the seed particles is a heat-activated polymerisation
initiator, polymerisation may be initiated by raising the
temperature of the reaction mixture to at least the activation
temperature of the initiator; for example, when dioctanoylperoxide
is selected as the organic compound, polymerisation may be
initiated by raising the temperature above 30.degree. C., typically
to 60 to 70.degree. C.
[0236] In embodiments, the Ugelstad process therefore
comprises:
[0237] forming an aqueous dispersion comprising the seed particles
and an emulsion comprising a water insoluble heat-activated
polymerisation initiator and a water-miscible organic solvent in
which the initiator is soluble and allowing the initiator to
diffuse into the seed particles;
[0238] contacting the particles with an aqueous medium comprising a
monomer and a cross-linker and allowing the monomer and the
cross-linker to diffuse into the polymer particles to form swollen
particles; and
[0239] heating the particles to activate the polymerisation
initiator and polymerise the monomer and the cross-linker within
the swollen particle, optionally wherein the monomer comprises an
acrylic monomer, a styrene monomer or a methacrylate monomer.
[0240] A porogen is included in the aqueous medium when porous
particles are to be made.
[0241] The monomer is ethylenically unsaturated and may be vinylic,
for example a styrenic monomer or an acrylic monomer. Styrenic
monomers may be mentioned in particular. Suitable monomers include
styrene, methyl methacrylate, methacrylic acid, hydroxyethyl
methacrylate, butylmethacrylate and acrylic acid and other acrylic,
e.g. methacrylic, monomers. The monomer may comprise a mixture of
monomer compounds, i.e. comonomers may be used. Further compounds
to be mentioned as monomers or comonomers are ethyl vinyl benzene,
vinyl pyridine, aminostyrene, methylstyrene,
2-hydroxyethylmethacrylate, methyl methacrylate, glycidyl
methacrylate, vinyl benzyl chloride, vinylchloride,
dimethylstyrene, ethylstyrene, ethylmethyl-styrene,
p-chlorostyrene, 2,4-dichlorostyrene, methyl acrylate, ethyl
acrylate, butylacrylate, methacrylic acid, ethyl
methylmethacrylate, maleic acid, maleic anhydride, dimethyl
maleate, diethyl maleate, dibutyl maleate, fumaric acid, dimethyl
fumarate, diethyl fumarate and acrylonitrile
[0242] The cross-linker may be a divinylic monomer, for example
divinylbenzene (DVB) or a di or multifunctional acrylate or
methacrylate, for example ethylene dimethacrylate, (EDMA).
Commercially available DVB is typically in admixture with a
significant proportion of ethylvinylbenzene (EVB) and in practice a
styrene/DVB mixture will therefore typically contain also EVB.
[0243] Functional groups may be introduced by the use of a
functionalised monomer or comonomer, e.g. glycidyl methacrylate,
HEMA (2-hydroxyethyl methacrylate), nitrostyrene or aminostyrene.
As an alternative to the polymer particles being formed carrying
surface functionalisation, or additionally thereto,
functionalisation of the polymeric material may take place after
polymerisation by, for example, nitration and subsequent reduction
of the thus-formed nitro groups to pendant amine groups, or direct
amination, for example by treatment with aminoethanol. After
preparation of functionalised polymer particles, functional groups
may be subjected to one or more functional group transformations,
for example nitro groups may be reduced to amino groups. Functional
groups, whether introduced in the synthesis of the particles, added
after synthesis of the particles, or created by transformation of
either such group, may undergo further reactions, for example to
conjugate the particles to another substance.
[0244] The present invention thus relates also to conjugates
comprising a particle of the invention coupled to another
substance, for example a ligand, by a residue of a reaction between
two functional groups. Suitable ligands include a biological
molecule, such as an antibody, an antibody fragment, a protein, a
polypeptide, an enzyme, a polynucleotide, biotin, a probe, a
primer, or a nucleic acid fragment; or chemical molecules, such as
chemical polymers, medicinal substances, cage molecules, chelating
agents, or catalysts. The present invention also relates to the
uses of these conjugates, for example use in biological assays.
[0245] One class of particles of the invention is non-magnetic.
Another class of particles is magnetic, e.g. superparamagnetic.
Suitable processes for preparing magnetic polymer particles are
described in U.S. Pat. No. 4,654,267 (Ugelstad) the contents of
which are incorporated herein by reference. U.S. Pat. No. 4,654,267
proposed a preparative method whereby, in its simplest form, porous
polymer particles are impregnated with solutions of iron compounds
whereafter the iron is precipitated, for instance by raising the pH
value. The precipitated iron compounds may then be converted to
superparamagnetic iron oxide crystals by heating. In the process,
solutions of iron salts and optionally salts of other metals which
may form magnetic ferrites, in water or in a mixture of water and
water-soluble organic solvents or in organic solvents, are mixed
with polymer particles in dry form or dispersed in water or in a
mixture of water and water-soluble organic liquids or in organic
liquids, and the metals are precipitated in the form of hydroxides,
for instance by raising the pH value, and, if desired, the
particles are heated.
[0246] To produce porous magnetic polymer particles having magnetic
material disposed within the polymer pores, U.S. Pat. No. 4,654,267
advocated the use of porous polymer particles having surface
functional groups which serve to draw the iron ions into the
polymer particles. U.S. Pat. No. 4,654,267 describes that examples
of monomers which had been found to be particularly suitable were
dimethylamino-ethylmethacrylate,
N-(dimethylaminopropyl)-methacrylic amide and vinyl pyridine, which
compounds provide functional groups which will bind the iron salts
with coordinate bonding. Other examples of suitable monomers
described in the US patent are those which contain ethylene oxide
groups or alkylene imine groups (--CH.sub.2--CHR--NH--, in which
R.dbd.H or alkyl).
[0247] U.S. Pat. No. 4,654,267 describes that it is also possible
to bind the iron by means of ionic bonds. By having acid groups on
and inside the particles, the iron may be transported from the
outer phase of the dissolved iron salt to be bound to these groups.
Examples of monomers which will provide such acid groups are
methacrylic acid, p-vinyl benzoic acid and maleic anhydride. It
will also be appreciated that acid groups can be created on
polymers made from monomers that lack native acid groups, e.g.
polystyrene, for example by reacting the polymer particles with a
mixture of sulphuric and nitric acid, to generate nitrated polymer
particles. The iron salt-binding groups may also be attached to the
premade polymers. Thus, it is possible to prepare a copolymer from
a monomer mixture which essentially consists of vinyl monomer with
epoxy group(s) such as glycidyl methacrylate. By treating the final
polymer with substances such as, for example, ethylenediamine which
react with epoxy groups and which contain N-- groups, said groups
will become covalently bonded on and inside the particles.
[0248] Where the porous polymer particles comprise nitrated polymer
particles, e.g. nitrated polystyrene, magnetic polymer particles
can be prepared by the following procedure. A solution comprising
the nitrated polymer particles and iron (II), e.g. FeSO.sub.4, is
made. After the polymer particles are impregnated with the iron
compound, the pH is increased, e.g. by addition of ammonia. This
causes partial oxidation of iron (II) to iron (III), partial
reduction of nitro groups of the polymer particles to amine groups
and precipitation of the iron ions as superparamagnetic ferrites,
e.g. maghemite and/or magnetite, in the pores of the polymer
particles. The amine groups provide surface functional groups that
can react with monomers used to form a coating polymer.
[0249] The leaching of magnetic crystals (e.g. superparamagnetic
crystals) from the porous polymer particles may be further
inhibited by forming a coating over the magnetic crystal-loaded
polymer particles. For example, a coating comprising at least one
transition metal oxide, e.g. a titanium oxide or a zirconium oxide,
can be formed over the superparamagnetic crystal-loaded polymer
particles, as described in WO 2008/079905 (incorporated herein by
reference in its entirety). Another exemplary coating is a polymer
coating, as described below.
[0250] The leaching of superparamagnetic crystals from the porous
polymer particles may be further inhibited by forming a coating
over the superparamagnetic crystal-loaded polymer particles, or
more particularly by at least partly filling the pores of the
particles with a polymer coating, as described in WO 2004/053490 or
WO 2006/075185 (incorporated herein by reference in their
entirety). The resultant particles comprise a matrix polymer,
magnetic crystals (e.g. superparamagnetic crystals) and a polymer
coating.
[0251] Such coating polymers may typically be formed from monomers
reactive with functional groups pendant from the surface of the
polymer of the underlying particles.
[0252] The coating polymer may be formed from at least one epoxide
compound, e.g. at least two epoxide compounds. The reaction of the
porous magnetic polymer particle with the coating monomers
generates a coating polymer within the pores of the matrix polymer
particles which serves essentially to block these pores, physically
encapsulating the superparamagnetic crystals within the polymer
particles. The resulting "coated" particles then have reduced
porosity relative to the porous starting material. It is believed
that the superparamagnetic crystals appear to catalyse the
polymerisation so that the coating forms preferentially in their
vicinity. Since the majority of the superparamagnetic crystals are
within pores in the starting porous particles, the coating may not
form to any significant extent on the external surface of the
particles.
[0253] In one embodiment, the porous polymer particles are reacted
with a mixture of epoxides.
[0254] The coating polymer may be formed from one or more epoxides.
At least one epoxide may contain at least one ether link and
optionally a hydrophobic component, e.g. an alkylene chain.
Generally the at least one epoxide will have a carbon atom content
of from 3 to 50, preferably 3 to 25. Typical epoxides that may be
used include epichlorohydrin, epibromohydrin, isopropylglycidyl
ether, butyl glycidyl ether, allylglycidyl ether, 1,4-butanediol
diglycidyl ether (1,4-bis(2,3-epoxypropoxy) butane),
neopentylglycol diglycidyl ether, ethylene glycol diglycidyl ether,
glycerol diglycidyl ether, glycidol, and glycidyl methacrylate,
ethyl hexyl glycidylether, methyl glycidylether, glycerol
propoxylate triglycidylether, poly(propylene glycol)
diclycidylether, 1,3 butanediol diglycidylether, tert-butyl
glycidylether, 1,4 cyclohexanedimethanol diglycidyl ether,
diethylene glycol diglycidyl ether, dodecyl glycidylether, O-(2,3
epoxypropyl)-0'-methylpolyethylene glycol glycidylether, glycidyl
tetrafluoroethyl ether, 1,6 hexanediol diglycidylether, octyl
glycidylether, decyl glycidylether,
poly(epichlorohydrin-co-ethylene oxide-co-allyl glycidylether),
polyethylene glycol diglycidyl ether, trimethylolethane
triglycidylether, trimethylolpropane, triglycidylether,
tert-butyldimethylsilyl glycidylether, 1,2-epoxybutane,
1,2-epoxypentane, 1,2-epoxy-5-hexene, 1,2-epoxy-hexane,
1,2-epoxy-7-octene, 1,2-epoxyoctane, 1,2,7,8-diepoxyoctane,
1,2-epoxy-9-decene, 1,2-epoxydecane, 1,2-epoxydodecane,
1,2-epoxytetradecane etc. In embodiments, the coating polymer is
formed from two or more epoxides which all contain at least one
ether link and optionally a hydrophobic component.
[0255] Typically, the coating reaction may be effected by
impregnating the porous magnetic polymer particle with the coating
monomers e.g. using a solution of these (for example in an organic
solvent such as methanol, toluene, xylene, diethyleneglycol,
dimethyl ether or diglyme) or by mixing a dispersion of the porous
particles in an organic solvent with a liquid epoxide mixture.
[0256] If desired, autofluorescence of the polymer particles may
reduced or avoided by keeping the particle essentially free of
conjugated delocalized electron systems, other than those in
benzene rings, as described in WO 2004/053490 (incorporated herein
by reference in its entirety). Such particles will not be
cross-linked with divinylbenzene, since any unreacted compound will
autofluoresce.
[0257] The polymer particles may be silica coated. The skilled
reader will require no explanation as to the formation of silica
coatings but a short summary is presented here for the non-skilled
reader. Silica coatings on polymer particles can be formed by the
same processes used for the polymerisation of metal alkoxides, for
example the sol-gel process. This is a process in which the
precursor, typically tertraethoxide silane (TEOS) undergoes a
series of hydrolysis and polycondensation steps, leading to the
silica coating on the polymer particles. In the first step, the
silica alkoxide precursor is partially hydrolysed. The degree of
hydrolysis is determined by the amount of water and catalyst (acid,
base) present. In a second step, partially hydrolysed molecules
react together in a condensation reaction, liberating small
molecules, typically water or alcohol. This continues and yields a
silica polymer. The structure of the silica polymer will be
influenced by the rate of hydrolysis and condensation, and thereby
catalysis. Prior to the silica coating the polymer particles can be
surf ace-functionalized. This provides a template capable of
reacting with the silicon alkoxide or the partially hydrolyzed
reaction products.
[0258] The stages of the method are illustrated below:
[0259] Hydrolysis:
.ident.Si--OR+H.sub.2O.ident.Si--OH+ROH
[0260] Alcohol Condensation:
.ident.Si--OR+.ident.Si--OH.ident.Si--O--Si.ident.+ROH
[0261] Water Condensation:
.ident.Si--OH+.ident.Si--OH.ident.Si--O--Si.ident.+H.sub.2O
[0262] The skilled reader will be aware that alternative
technologies exist for applying silica coatings, e.g. water-in-oil
emulsion techniques. For more information, the reader is referred
to Brinker C. J. and Scherer G., SOL-GEL SCIENCE: The Physics and
Chemistry of Sol-Gel Processing, Academic Press, San Diego 1990;
and to Ziegler J. M. and Fearon F. W., Silicon-Based Polymer
Science: A Comprehensive Resource, American Chemical Society,
Washington D.C. 1990.
[0263] The swelling and polymerisation stages are typically
performed in aqueous dispersion in the presence of materials, e.g.
surfactants, stabilizers, organic solvents, etc., which it is
desirable to remove from the particles. Likewise, it may be
desirable to remove linear polymers which formed the seed
particles, for example to avoid leakage during use in
chromatography. Generally a water-miscible organic solvent in which
the cross-linked polymer is insoluble, or an aqueous solution of
such a solvent, may be used for removal of contaminants and linear
polymers. However it is particularly suitable to use butyl acetate
in this regard in view of its effectiveness in removing undesired
residues from the Ugelstad polymerisation process.
[0264] Depending on their desired end use, the monodisperse polymer
particles may be coated (e.g. with metallic coatings); they may
have materials, e.g. magnetic crystals, specific binding partners
(e.g. antibodies, avidin or streptavidin, etc.), or catalysts bound
to their surface or deposited in pores or on the surface; or they
may be expanded (e.g. using blowing agents). The invention
therefore includes monodisperse submicron polymer particles as
described herein having a matrix polymer (e.g. polystyrene) which
may be porous and optionally further having one or more additional
substances, for example selected from magnetic material included in
any pores, one or more coating materials, one or more functional
groups, one of more conjugated substances (e.g. specific binding
partners, nucleic acids, proteins, other biological molecules or
structures). The particles may be coupled to a substrate.
[0265] The invention includes particles obtained by, or having the
characteristics of particles obtained by, the preparative processes
described herein.
[0266] The Ugelstad processes described herein may be worked to be
highly reproducible and scaleable. The invention therefore enables
consistency between and within batches, which is a prerequisite for
industrial application. The invention also enables production of
pilot scale batches of e.g. at least 300 g as well as kilogram
scale industrial batches, which is another prerequisite for viable
industrial production. In contrast, suspension polymerisation
processes seem to suffer from a lack of reproducibility and
scaleability which makes them non-viable for industrial use, to the
extent that one batch of particles may lack good and consistent
spherical particle shape. The invention therefore provides the
following inventions in relation to the particles and manufacturing
processes of the disclosure: [0267] manufacturing processes which
result in batches of particles wherein the weight of particles when
determined as dry particles is at least 300 g, e.g. at least 500 g
and optionally at least 1 kg, for example at least 5 kg as in the
case of at least 10 kg [0268] a particle batch wherein the weight
of particles when determined as dry particles is at least 300 g,
e.g. at least 500 g and optionally at least 1 kg, for example at
least 5 kg as in the case of at least 10 kg [0269] a particle batch
wherein the weight of particles when determined as dry particles is
at least 300 g, e.g. at least 500 g and optionally at least 1 kg,
for example at least 5 kg as in the case of at least 10 kg and
wherein the particles of any two or more sub-populations (e.g. of
at least 100 and optionally at least 1000 particles) of the batch
have substantially indistinguishable characteristics, e.g. when 2,
5, 10 or 20 sub-populations (i.e. samples) are selected, optionally
wherein the indistinguishable characteristics include one or a
combination of (e.g. all of) size, shape, surface morphology,
swelling properties and specific surface area [0270] methods
comprising the parallel or sequential performance of batch
manufacturing processes to result in 2 or more batches of polymer
particles, for example 5 or more batches, e.g. 10 or more batches,
wherein the particles of each batch have identical characteristics
with the particles of each other batch within industrial acceptable
tolerance, e.g. the variation between batches of one or more of
(e.g. all of) size, swelling properties and specific surface are
15% or less, e.g. 10% or less and optionally 5% or less, wherein
the weight of particles of each batch when determined as dry
particles may for example be at least 300 g, e.g. at least 500 g
and optionally at least 1 kg, for example at least 5 kg as in the
case of at least 10 kg [0271] a collection of 2 or more batches of
polymer particles, for example 5 or more batches, e.g. 10 or more
batches, wherein the particles of each batch have identical
characteristics with the particles of each other batch within
industrial acceptable tolerance, e.g. the variation between batches
of one or more of (e.g. all of) size, swelling properties and
specific surface are 15% or less, e.g. 10% or less and optionally
5% or less, wherein the weight of particles of each batch when
determined as dry particles may for example be at least 300 g, e.g.
at least 500 g and optionally at least 1 kg, for example at least 5
kg as in the case of at least 10 kg [0272] a method for the
delivery of particles comprising transporting on or in a vehicle
(e.g. a road vehicle, a ship or an aircraft) at least one batch
(for example 5 or more batches, e.g. 10 or more batches) of
particles wherein the weight of particles of each batch when
determined as dry particles is at least 300 g, e.g. at least 500 g
and optionally at least 1 kg, for example at least 5 kg as in the
case of at least 10 kg.
[0273] In view of the consistency of the quality and
characteristics which the particles of the disclosure may possess,
they may be used in methods which comprise performing processes in
relation to a conjugated substance, e.g. selected from labels,
biological molecules and biological structures, for example
biological molecules such as amino acids, saccharides, nucleotides
and nucleosides and multimers made by condensing together two or
more such monomers, e.g. polypeptides, proteins, polysaccharides,
oligonucleotides and nucleic acids. As labels may be mentioned
dyes, e.g. fluorescent dyes, quenchers, enzymes, and semiconductor
nanocrystals. The invention includes such uses as well as: [0274]
i) conjugates comprising a population of particles of the
disclosure at least a portion of which are coupled to a conjugated
substance, e.g. one as just described [0275] ii) a method
comprising coupling at least a portion of a population of particles
of the disclosure to a substance, e.g. one as just described [0276]
iii) a method comprising coupling at least a portion of a
population of particles of the disclosure to a substrate.
[0277] The Ugelstad processes described herein can be performed
consistently without the problems which in practice can arise with
emulsion polymerisation, e.g. agglomeration of particles as well as
variation in the product.
Uses of the Particles
[0278] The particles, whether magnetic or non-magnetic, can be used
in many applications, e.g. information storage, color imaging,
bioprocessing, diagnostic microbiology, biosensors and drug
delivery. Magnetic particles may be used in magnetic refrigeration,
ferrofluids and magnetic switches. In particular, the magnetic
particles, for example magnetic particles coupled to a ligand or
magnetic particles comprising one or more specific binding
partners, can be detected by magnetic detectors, for example a
giant magnetoresistive sensor (GMR), Hall sensor, or
superconducting quantum interference device (SQUID) sensor.
[0279] A SQUID is a very sensitive magnetometer that can be used to
measure extremely small magnetic fields, based upon superconducting
loops containing Josephson junctions. Magnetic particles of the
invention typically have small magnetic fields, e.g. due to the
small size of the particles. The high sensitivity of SQUID means
that it is a particularly suitable detector for the magnetic
particles of the invention.
[0280] The magnetic particles can, for example, be used as contrast
agents in SQUID imaging, as described in EP0523116 (incorporated
herein by reference in its entirety). The magnetic particles can
also be used in an assay, e.g. an affinity based assay and/or a
bioassay and/or a competitive binding assay. In this regard it will
be appreciated that the magnetic particles of the invention can be
used as a detection tag in the assay, linked to one of the binding
partners in an assay. For example, a specific binding partner (for
instance an affinity molecule), e.g. an antibody, can be attached
to the surface of a magnetic particle. Where the specific binding
partner is an antibody, an antigen-antibody reaction will take
place between the antibody and an antigen (the target substance) to
produce a weak magnetic field signal attributable to the magnetic
marker, which can be measured by a magnetic detector, e.g. a SQUID,
as is described in US 2006/0035388 (incorporated herein by
reference in its entirety).
[0281] The particles, e.g. functionalised polymer particles, can be
used to assist in creating a semi-ordered array of molecules for
assaying. For example, such molecules may be bound to the particles
with a first binding site where binding may be covalent; the
binding is optionally cleaveable by enzymatic, chemical, photonic
or other appropriate methods. The molecules to be assayed can
contain a second binding site designed to bind to a third binding
site on a surface to which deposition will occur. In this sense,
the particles can act as spacers preventing two molecules from
binding to the surface at less than a user defined distance where
such distance is defined by the diameter of the particle. This can
be useful for individual molecules so that they may be individually
detectable by minimizing crosstalk from neighbouring molecules. It
can also be useful for depositing molecules, such as
polynucleotides (or RNA, or proteins, or other biomolecules) that
will be amplified. In embodiments using amplification, overlap of
the resulting localized populations can be controlled by
considering the size of the particles, the rate of amplification
and the amount of time for which the reaction is run. After initial
deposition, or after amplification, the particles can be cleaved
granting easier access to the molecules to be assayed. Methods
involving the use of silica particles to assist in creating a
semi-ordered array of molecules are disclosed in J. J. Schwartz and
S. R. Quake, "High density single molecule surface patterning with
colloidal epitaxy", Applied Physics Letters, 91, 083902 (2007) and
US 2009/0053690 (both incorporated herein by reference in their
entirety) and it will be appreciated that these methods can be
readily adapted to particles of the present invention, e.g. polymer
particles functionalised with amino groups.
[0282] Silica-coated particles may be useful in processes for the
analysis or treatment of nucleic acids the well known principle of
nucleic acid binding to a silica surface. U.S. Pat. No. 5,234,809,
for example, describes a method where nucleic acids are bound to a
solid phase in the form of silica particles, in the presence of a
chaotropic agent such as a guanidinium salt, and thereby separated
from the remainder of the sample. Processes using magnetic
particles are increasingly being used as high-throughput techniques
for the automated isolation of nucleic acids, in which total
nucleic acid (both DNA and RNA) is isolated from a biological
sample by reversible binding to SiOH-modified magnetic particles.
For this purpose the nucleic acids to be isolated are contacted
with silica-modified magnetic particles in a chaotropic binding
buffer. In a typical process using silica-coated particles, the
binding of the nucleic acids to the particle surface takes place
over a range of temperatures, from .about.18.degree. to
.about.38.degree. C. for example, over a period of time up to an
hour while the particle suspension is mixed by shaking or
vortexing. The particles loaded with nucleic acids are then drawn
towards the vessel wall by applying a magnetic field, and the
supernatant is aspirated and discarded. After removing the magnetic
field, the particles are resuspended and washed several times with
a washing buffer or buffers. The nucleic acids bound to the
magnetic particles are then removed from the particles at a high
temperature, such as for example at 90.degree. C. for 10 mins, with
the aid of an elution buffer. After re-applying the magnetic field,
the eluate containing the nucleic acids can be pipetted off.
[0283] The SOLiD.TM. sequencing system (Sequencing by
Oligonucleotide Ligation and Detection) of Applied Biosystems uses
stepwise cycled ligation for high throughput DNA sequencing. In
this bead based system, beads (i.e. polymer particles) loaded with
DNA templates undergo sequential ligation and cleavage reactions
using 4-colour, fluorescently-labeled octameric probes. These
probes are delivered serially and serve to interrogate dinucleotide
positions on DNA strands. It would be desirable to support higher
bead densities that facilitate an increased number of bead events
per instrument run and improved probe chemistry, affording
increased sequencing fidelity.
[0284] Sequencing by Oligonucleotide Ligation and Detection
involves attachment of a nucleic acid target to a cross-linked
polymer particles (beads) followed by immobilization of a plurality
of the particles onto a surface. Each nucleic acid-bead conjugate
comprises a unique DNA sequence, Sequencing techniques of this type
are disclosed in International Publication No. WO 2006/084132 A2
(included herein by reference).
[0285] Methods of attachment of the beads to the support have
utilized a flat glass microscope slide irreversibly coated with
streptavidin. Nucleic acid-laden beads are contacted with
biotinylated nucleotides (e.g., obtained by the action of
biotinylated dNTP's and terminal deoxytransferase on the DNA target
subsequent to attachment to the bead). Incubation of the
biotinylated beads with the streptavidin coated slide results in
immobilization of the beads onto the slide by the interaction of
streptavidin with the biotin. While kinetically this is a very
effective attachment scheme, movement of the beads on the slide was
sometimes observed under the conditions required by the DNA
sequence assay. When beads are present in high densities on the
slide (e.g., up to 100,000 beads/mm.sup.2) and interrogated
multiple times (e.g., up to 25 times), any significant bead
movement can preclude robust identification of a particular bead on
subsequent scans within a dense population of beads.
[0286] US 2009/0099027 (equivalent to WO2009026546, both included
herein by reference) therefore describes a covalent system for bead
immobilization that reduces movement of the beads during sequencing
and other forms of genetic analysis. The method comprises: reacting
a nucleophilic group on the surface of a substrate with a molecule
comprising a plurality of electrophilic groups thereby providing
one or more free electrophilic groups on the surface of the
substrate; and reacting nucleophilic groups on a surface of a
particulate material with the one or more free electrophilic groups
on the surface of the substrate to covalently attach the
particulate material to the substrate.
[0287] US 2009/0099027 describes the modification of a nucleophilic
(more particularly, amino functional) surface with a
multifunctional electrophilic reagent. For example, the
electrophilic surfaces of silicate glass microscope slides can be
readily converted to a nucleophilic surface by reacting surface
groups with (aminopropyl) trialkoxysilanes.
[0288] A DNA target nucleic acid that had been covalently attached
to a cross-linked polymer bead may be modified by the action of
aminoalkyl dNTP's and terminal deoxytransferase on the DNA target
subsequent to attachment to the bead. The nucleophilic amino group
on the DNA target can then react with the residual electrophilic
group of the support surface to form multiple stable covalent bonds
between the bead and the glass surface.
[0289] It has been found that stable covalent bonds can be formed
between a surface containing electrophilic groups and particles
containing nucleophilic groups. In addition, beads containing
nucleophilic amino groups from the action of amino-dNTP's and
terminal deoxytransferase on a DNA target can be immobilized under
aqueous basic conditions on the modified surface. For example,
surfaces comprising amino groups that have been activated with
benzene 1,4-diisothiocyanate can be used to immobilize beads with
nucleophilic groups. In addition, the covalent attachment appears
to be quite stable, and no bead movement is observed.
[0290] The surface immobilized beads can be used in methods of
analysing nucleic acid sequences based on repeated cycles of duplex
extension along a single stranded template via ligation. Sequencing
methods of this type are disclosed in U.S. Pat. Nos. 5,750,341;
5,969,119; and 6,306,597 B1 and in International Publication No. WO
2006/084132 A2. Each of these publications is incorporated by
reference herein in its entirety. Moreover, the techniques
described in the aforementioned publications can be used to analyse
(e.g., sequence) nucleic acid templates attached to particles that
are bound to supports as described herein. The immobilized beads
can be used in sequencing methods that do not necessarily employ a
ligation step, such as sequencing using labeled nucleotide that
have removable blocking groups that prevent polynucleotide chain
extension (e.g., U.S. Pat. Nos. 6,664,079; 6,232,465; and
7,057,026, each of which is incorporated by reference herein in its
entirety). The immobilized beads can be used in a variety of
techniques in which signals on the beads are repeated detected
through multiple cycles.
[0291] The beads which are used in SOLiD sequencing may be
monodisperse submicron particles of the disclosure. The present
invention therefore includes the use of the monodisperse submicron
particles in the methods and products disclosed in the publications
mentioned in the previous paragraph and the applicant of the
present application considers all such uses, methods and products
to fall within the present invention and reserves the right to
claim them. The use of submicron particles in SOLiD sequencing
enables a greater density of particles to be attached to the glass
surfaces (e.g. glass panels or microscope slides). Further included
in the present invention is a method of performing SOLiD sequencing
which uses monodisperse submicron particles of the disclosure, e.g.
wherein monodisperse submicron particles of the present disclosure
are coupled to a nucleic acid target and immobilised on a surface,
e.g. a glass surface. The method of immobilisation is not critical
and may be covalent or non-covalent, examples of non-covalent
coupling being through streptavidin/avidin-biotin binding. The
covalent coupling may be as described in US 2009/0099027 and
WO2009026546, for example, but any other suitable technique for
covalent coupling may be used. Included in the invention,
therefore, is a method of forming a product (an article of
manufacture), comprising coupling monodisperse submicron particles
of the present disclosure to a nucleic acid and optionally further
comprising immobilising the resultant nucleic acid-laden particles
on a surface, e.g. a glass surface. The nucleic acid may be used as
a target in sequencing, e.g. using SOLiD sequencing.
[0292] For example, a method is provided that comprises:
[0293] (a) hybridizing a first initializing oligonucleotide probe
to a target polynucleotide to form a probe-target duplex, wherein
the oligonucleotide probe has an extendable probe terminus, wherein
the target polynucleotide is attached to a polymer particle which
is a member of a population of polymer particles as disclosed
herein and wherein the particle is covalently attached to the
surface of a solid support;
[0294] (b) ligating a first end of an extension oligonucleotide
probe to the extendable probe terminus thereby forming an extended
duplex containing an extended oligonucleotide probe, wherein the
extension oligonucleotide probe comprises a cleavage site and a
detectable label;
[0295] (c) identifying one or more nucleotides in the target
polynucleotide by detecting the label attached to the just-ligated
extension oligonucleotide probe;
[0296] (d) cleaving the just-ligated extension oligonucleotide
probe at the cleavage site to generate the extendable probe
terminus, wherein cleavage removes a portion of the just-ligated
extension oligonucleotide probe that comprises the label from the
probe-target duplex; and
[0297] (e) repeating steps (b), (c) and (d) until a sequence of
nucleotides in the target polynucleotide is determined.
[0298] Also provided is a method of sequencing a nucleic acid
comprising:
(a) hybridizing a primer to a target polynucleotide to form a
primer-target duplex, wherein the target polynucleotide is attached
at a 5' end to a polymer particle which is a member of a population
of polymer particles as disclosed herein and wherein the polymer
particle is covalently attached to the surface of a support; (b)
contacting the primer-target duplex with a polymerase and one or
more different nucleotide analogues to incorporate a nucleotide
analogue onto the 3' end of the primer thereby forming an extended
primer strand, wherein the incorporated nucleotide analogue
terminates the polymerase reaction and wherein each of the one or
more nucleotide analogues comprises (i) a base selected from the
group consisting of adenine, guanine, cytosine, thymine and uracil
and their analogues (ii) a unique label attached to the base or
analogue thereof via a cleavable linker; (iii) a deoxyribose; and
(iv) a cleavable chemical group which caps an --OH group at a
3'-position of the deoxyribose; (c) washing the surface of the
support to remove any unincorporated nucleotide analogues; (d)
detecting the unique label attached to the just-incorporated
nucleotide analogue to thereby identify the just-incorporated
nucleotide analogue; (e) optionally, permanently capping any
unreacted --OH group on the extended primer strand; (f) cleaving
the cleavable linker between the just incorporated nucleotide
analogue and the unique label; (g) cleaving the chemical group
capping the --OH group at the 3'-position of the deoxyribose of the
just incorporated nucleotide analogue to uncap the --OH group; (h)
washing the surface of the support to remove cleaved compounds; (i)
repeating steps (b)-(h).
[0299] The polymer particles of the disclosure may be used in any
method of nucleic acid sequencing which involves a polymer
particle. The invention includes particles of the disclosure
coupled to a nucleic acid as well as a method of sequencing a
nucleic acid which comprises coupling a nucleic acid to a
population of particles of the disclosure. The nucleic acid may be
DNA or RNA.
[0300] The present disclosure includes a product (e.g. an article
of manufacture) comprising a plurality of monodisperse submicron
particles of the disclosure coupled to a substrate such as, for
example, glass surface, for example through a streptavidin-biotin
linkage, an avidin-biotin linkage or through a covalent linkage,
e.g. as described in US 2009/0099027 and WO2009026546. The
particles may be coupled to the substrate through a nucleic acid.
The present disclosure includes the use of the monodisperse
submicron particles of the disclosure to make such a product. The
present invention includes the use of the attachment chemistry
described in US 2009/0099027 and WO2009026546 to attach
monodisperse submicron particles of the disclosure to a substrate,
and the applicant reserves the right to claim methods of using such
chemistry and the products thereof. The present specification
therefore includes by reference the disclosures of US 2009/0099027
and WO2009026546, including without limitation [0007] to [0029],
[0057] to [0094] and the claims of US 2009/0099027 and the
applicant reserves the right both to claim combinations of such
teachings with monodisperse submicron beads of the present
disclosure (i.e. in which the particles/beads of the US
specification are replaced by particles of the present disclosure)
and to reproduce the contents of US 2009/0099027 and WO2009026546,
including without limitation [0007] to [0029], [0057] to [0094] of
US 2009/0099027, verbatim in the present specification. For the
avoidance of doubt, it is hereby confirmed that the applicant
reserves the right to reproduce in the present specification the
figures of US 2009/0099027 referred to in said paragraphs, and
incorporates said figures herein by reference.
[0301] The invention therefore includes methods in which
functionalised monodisperse polymer particles of the disclosure are
subjected to one or more further reactions to obtain a desired
product. The invention also includes the use of these products in
applications.
Analytical Methods
Molecular Weight Measurement
[0302] The molecular weight distribution of the polymers in a seed
particle or other non cross-linked polymeric particle can be
measured by a form of size exclusion chromatography (SEC), e.g. gel
permeation chromatography (GPC), calibrated with suitable polymeric
molecular weight standards. For example, determination of the
molecular weight of polystyrene polymers by GPC ideally uses
polystyrene molecular weight markers as set out in the following
procedure. A calibration curve, e.g. an 8 point calibration curve,
is prepared using polystyrene standards; (Polymer Labs) PS-1 MW
range 266-8,000,000 amu can be used. The sample is dissolved in
tetrahydrofuran (THF) containing 0.015% sulphur (S added as a
retention time marker) to make a solution of 0.5 mg/ml, and
filtered (0.45 .mu.m Nylon Aerodisc) prior to instrumental
analysis. Both the standards and the sample are suitably run on the
SEC instrumentation detailed below, allowing molecular weight to be
determined.
Instrumentation:
[0303] The SEC system used for GPC may consist of the following
units:
Rheodyne i725 injector with 100 .mu.l sample loop Waters 510 HPLC
pump Waters 484 Tunable absorbance (UV) Detector operated at 254
nm
Column Set:
Pre-Column Filter
[0304] 2. PLgel 5 .mu.m Mixed C Waters (in THF) [0305] connected in
series and placed in a column heating module Waters 038040 Column
temperature. 40.degree. C. Eluent: THF (HPLC grade) The THF has
been pre-filtered (Millipore Fluoropore 0.45 .mu.m membrane
filter).
Size and Size Distribution
[0306] The size distribution of samples can be measured using disc
centrifugation, e.g. CPS Disc Centrifugation.TM. on Disc Centrifuge
Model DC20000, using protocols provided by the instrument
manufacturer. Accurate results require calibration with a standard
of similar density to the sample being analysed and thus is only of
use where a suitable polymeric standard is available, for example a
set of compact polystyrene particle standards for particles of the
disclosure comprising predominantly polystyrene. Where the samples
being measured have a density that is not known, e.g. for porous
particles, the measurement obtained by CPS disc centrifugation will
be reproducible but will not provide the actual diameter.
[0307] Photon correlation spectroscopy (PCS) can be used to obtain
the hydrodynamic diameter of a particle in the form of the
z-average. The measurement is independent of the particle density
and based on Brownian motion of small particles. PCS measurements
for nanosized particles can be obtained, for example with a Malvern
ZetaSizer Nano-ZS, Model ZEN3600. Further details and methods can
be found in the Malvern Zetasizer Nano series manual (incorporated
herein by reference in its entirety).
[0308] Another technique that can be used to determine the
diameters of individual submicron particles is measurement of the
diameter of the dry polymer particles as imaged by scanning
electron microscopy (SEM). Dry particle samples can be prepared for
SEM imaging by capture on an SEM compatible surface and coating of
the sample with carbon or gold by vapour deposition. The diameter
can be determined by individual measurements of the particles
appearing the SEM image. When assessing the surface morphology of
submicron particles, e.g. of at least 200 nm and less than 1000 nm
diameter, a suitable SEM magnification is 10,000. Measurements
should be made of at least 10 particles. SEM images can, for
example, be obtained with a Philips XL30 instrument, operated at an
acceleration voltage of 20 kV, with a typical detection area of
0.0004 mm.sup.2 and magnification of 10,000.
[0309] PCS is a preferred method of determining average diameter,
suitable for use with particles of both known and unknown
density.
Visual Appearance
[0310] Visual appearance of SEM images is particularly important
for characterizing the surface morphology of the particles, e.g.
whether the particles have a relatively smooth surface (regular
spherical shape) or are rough and irregular (so-called "cauliflower
particles"). Polymer particles of the invention have a smooth,
spherical appearance at a magnification of 10,000. As mentioned
above, measurements made from an SEM image can also be used to
determine the size of individual particles and the size
distributions of relatively small populations, such as for 20 to 50
particles.
Surface Area and Pore Size Distribution
[0311] Surface area can be measured by gas adsorption methods, with
the surface area calculated using BET theory (see, e.g. Chapter 3,
"Surface area and pore structure by gas adsorption" of P A Webb and
C Orr, Analytical methods in fine particle technology,
Micromeritics, 1997, incorporated herein by reference in its
entirety). An example of an instrument that can be used to perform
surface area measurements of submicron beads is the Tristar Surface
Analyser and Porosity Analyser. This instrument can be used to
measure the specific surface area and also the pore size
distribution. When measuring the pore size distribution of polymer
particles, the procedure measures the distribution of small pores
with a pore radius of from approximately 10 .ANG. to 350 .ANG.. The
determination of pore size distribution is based upon the BHJ
method and the Harkins-Jura equation to estimate the film
thickness.
Detection of Coatings
[0312] The presence of coatings on polymeric beads can be
determined in a number of ways.
[0313] Infrared spectroscopy (IR), for example fourier transform
infrared (FTIR) spectroscopy, can be used to qualitatively detect
the presence of functional groups or other coatings on the surface
of polymeric beads. Coating increases the mass of the beads, so
detection of a weight increase relative to uncoated beads is
indicative of the presence of a coating. Coated beads also
typically have a reduced surface area compared to uncoated beads,
so comparative surface area measurements can be used to confirm the
presence of a coating. There are also other methods that are
suitable for detecting the presence of specific functional groups,
for instance: [0314] titration to detect acidic functional groups,
e.g. to detect free carboxylic acid moieties, or [0315]
determination of amine groups on a polystyrene bead by ultraviolet
(UV) spectroscopy.
Characterisation of Crosslinking
[0316] A method for determining the level of cross-linking by
ascertaining the amount of swelling induced by solvent and
correlating this with known standards has previously been described
in this specification. For a more detailed discussion of
characterisation of crosslinking, the reader is referred to
Harrison, D J P, Yates, W R and Johnson, J F (1985) `TECHNIQUES FOR
THE ANALYSIS OF CROSSLINKED POLYMERS`, Polymer Reviews, 25:4,
481-549. Methods based on the swelling of polymers are described on
pages 494-504 of this publication. Harrison eta/describe a number
of techniques to measure swelling, both volumetric and
gravimetric.
[0317] The swellability of a polymer when contacted with a
particular solvent depends on the polymer class to which the
polymer belongs, e.g. the solubility of polystyrene and a
polyacrylate in a particular solvent may differ widely. It is
therefore necessary for the known particles used as standards to
determine swellability to belong to the same polymer class as the
test particle/particle population. The polymer class may be
determined by known analytical techniques, in particular mass
spectrometry, where pyrolysis mass spectrometry is especially
useful to determine polymer class of cross linked polymers. FTIR
and NMR may also help resolve the polymer class. The application of
mass spectrometry to polymer analysis is described in S. D. Hanton,
Mass Spectrometry of Polymers and Polymer Surfaces, Chem. Rev.
2001, 101, 527-569. A more detailed account of pyrolysis mass
spectrometry may be found in Kuangnan Qian, William E. Killinger,
and Melissa Casey, Analytical Rapid Polymer Identification by
In-Source Direct Pyrolysis Mass Spectrometry and Library Searching
Techniques, Anal. Chem. 1996, 68, 1019-1027.
[0318] The degree of cross-linking of an unknown polymer particle
population may therefore be determined by analysing a specimen
particle or particle group to determine the polymer class, and then
by comparing the swellability of the unknown particles in a solvent
with the swellability in the same solvent of a plurality of
standards belonging to the same polymer class.
[0319] Accordingly, by comparing the swelling of an unknown polymer
sample against comparable standards, the degree of cross-linking
(expressed as the amount of crosslinker monomer used in manufacture
as discussed above) may be determined or approximated.
[0320] The examples below are given so as to illustrate the
practice of this invention. They are not intended to limit or
define the entire scope of this invention. The reagents employed in
the embodiments below are commercially available or can be prepared
using commercially available instrumentation, methods, or reagents
known in the art. The examples illustrate various aspects of the
invention and practice of the methods of the invention. The
examples are not intended to provide an exhaustive description of
the many different embodiments of the invention. Thus, although the
foregoing invention is described in some detail by way of
illustration and example for purposes of clarity of understanding,
those of ordinary skill in the art will realize readily that many
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims.
EXAMPLES
Synthesis of Seed Particles
[0321] In embodiments, the seed is synthesised comprising monomer
(M), styrene; a water-soluble initiator (I), potassium persulfate
or ammonium persulfate; a surfactant (S), sodium dodecylsulfate
(SDS); optionally a buffer, borax; and a chain transferring agent
(T), bromotrichloro methane, alkyl mercaptans. The polymerisation
procedure is an emulsion polymerisation performed with a surfactant
below the critical micelle concentration. The seed synthesis
follows generally the same procedures for seeds formed by emulsion
polymerisation as that described in WO00/61647 (incorporated herein
by reference in its entirety). The difference in the comparable
procedures is: [0322] The type and amount of surfactant is changed.
SDS is used in the present procedure, with the concentration of SDS
varied between 1 and 2 g/L. In the examples given below, the SDS
used had a purity of .gtoreq.98.5%. [0323] The present synthesis
involves the addition of the chain transferring agent (T). The
chain transfer agent is added after the commencement of nucleation,
e.g., at a concentration of at least 1.5 mmol/L water and not more
than 45 mmol/L water. The occurrence of nucleation is detected by
the emulsion becoming cloudy, for example turning white when
styrene is used as the monomer.
Example 1
Synthesis of Low Molecular Weight Seed Particles
[0324] Preparation of Initial Seed Particles with a Weight Average
Molecular Weight of 1.8.times.10.sup.4 gram/mol and a Mode Diameter
of 0.12 .mu.m.
[0325] 84.6 g styrene was extracted with 150 ml 10 wt % sodium
hydroxide, then washed with water to pH7 and then flushed with
argon for 10 min. In a 2 L reactor, 1733 g of water and 0.59 g of
borax were heated to 80.degree. C., and 50 g water was evaporated
off under reduced pressure to remove oxygen. Then 2.25 g sodium
dodecyl sulfate (SDS) in 20 ml boiled water was added and the
mixture was stirred for 10 min, then the washed and substantially
oxygen free styrene was added and the mixture was stirred for a
further 15 min. 3.08 g ammonium persulfate in 107 ml boiled water
was then added. After 5 minutes the emulsion turned white. After an
additional 10 minutes 5.5 gram of 1-octanethiol was added. The
mixture was kept at 80.degree. C. in an argon atmosphere for 20
hours. A dispersion of monosized polymeric particles was formed
having a particle mode diameter of 0.12 .mu.m determined by disc
centrifugation, as illustrated in FIG. 3.
Example 2
Synthesis of Low Molecular Weight Seed Particles
[0326] Preparation of Initial Seed Particles with a Weight Average
Molecular Weight of 1.16.times.10.sup.4 gram/mol and a z Average
Diameter of 0.14 .mu.m.
[0327] 84.7 g styrene was extracted with 150 ml 10 wt % sodium
hydroxide, then washed with water to pH7 and then flushed with
argon for 10 min. In a 2 L reactor, 1720 g of water and 0.59 g of
borax were heated to 80.degree. C., and 50 g water was evaporated
off under reduced pressure to remove oxygen. Then 2.34 g sodium
dodecyl sulfate (SDS) in 20 ml boiled water was added and the
mixture was stirred for 10 min, then the washed and substantially
oxygen free styrene was added and the mixture was stirred for a
further 15 min. 3.08 g ammonium persulfate in 100 ml boiled water
was then added. After 5 minutes the emulsion turned white. After an
additional 10 minutes 2.38 mL bromotrichloromethane was
continuously added at a rate of 0.05 mL/min. The mixture was kept
at 80.degree. C. in an argon atmosphere for 12 hours. A
monodisperse suspension of polymeric particles was formed having a
z average diameter of 0.14 .mu.m determined by photon correlation
spectroscopy.
Example 3
Synthesis of Low Molecular Weight Seed Particles
[0328] Preparation of Initial Seed Particles with a Weight Average
Molecular Weight of 1.0.times.10.sup.4 gram/mol and a z Average
Diameter of 0.15 .mu.m.
[0329] 98.7 g styrene was extracted with 175 ml 10 wt % sodium
hydroxide, then washed with water to pH7 and then flushed with
argon for 10 min. In a 2 L reactor, 2000 g of water and 0.68 g of
borax were heated to 80.degree. C., and 50 g water was evaporated
off under reduced pressure to remove oxygen. Then 2.62 g sodium
dodecyl sulfate (SDS) in 20 ml boiled water was added and the
mixture was stirred for 10 min, then the washed and substantially
oxygen free styrene was added and the mixture was stirred for a
further 15 min. 3.59 g ammonium persulfate in 101 ml boiled water
was then added. After 7 minutes the emulsion turned white. After an
additional 10 minutes 6.4 g bromotrichloromethane was added. The
mixture was kept at 80.degree. C. in an argon atmosphere for 18
hours. A monodisperse suspension of polymeric particles was formed
having a z average diameter of 0.15 .mu.m determined by photon
correlation spectroscopy.
Application of the Ugelstad Process to Form Polymer Particles from
Seed Particles
[0330] Reaction scheme 1 illustrates, in general terms, an
embodiment of the Ugelstad process that can be used to create
submicron polymer particles. In reaction scheme 1, the seed
particle contains P, a low molecular weight polymer. Y is an
organic molecule with a suitably low water solubility, which may
also be a polymerisation initiator, for example dioctanoylperoxide.
The weight ratio Y/P is of the order of 1 to 3 after the activation
step. In the next step one or more monomers are added, for instance
the illustrated monomers 1,3-divinylbenzene and styrene, optionally
with a porogen, such as toluene. The weight ratio of (monomer plus
porogen) to polymer may be 60 to 100. After allowing the one or
more monomers to swell the activated seed particle, the monomers
are polymerised, forming the polymer particles.
##STR00001##
Example 4
Synthesis of Polymer Particles from Low Molecular Weight Seed
Particles
[0331] 17.8 g SDS, 1480 g water, 74 g acetone and 148.0 g
dioctanoylperoxide were mixed with an ultraturax type
Ystral.TM.X10/25 homogeniser ("ultraturax") for 3 minutes and
homogenised with a pressure homogeniser for 10 minutes (=initiator
emulsion).
[0332] 330.9 g toluene, 118.7 g 65% divinylbenzene (DVB)
(comprising 74.8 g DVB and 43.9 g ethylvinylbenzene), 30 g styrene,
44.5 g polyvinylpyrrolidone (PVP) K-30, 2.7 g SDS and 2827.0 g
water were mixed by ultraturax for 4 minutes, and further
homogenised for 30 minutes (=monomer emulsion).
[0333] In a 0.5 L reactor 23.3 g of the seed particle dispersion
prepared according to Example 1 was gently mixed with 25.9 g of the
initiator emulsion. The mixture was stirred at 25.degree. C. for 24
hours.
[0334] In a 0.5 L reactor 22.4 gram of the activated seed particles
and 344.3 gram of the monomer emulsion was added. The mixture was
stirred for 2 hours at 25.degree. C. and then 133.4 g water was
added, and the mixture was then heated to 60.degree. C. After 1
hour at 60.degree. C. the temperature was raised to 70.degree. C.
and maintained at this temperature for 20 hours.
[0335] The dispersion was repeatedly washed with methanol, then air
dried over night followed by a further 15 hours of drying at
50.degree. C.
[0336] FIG. 4 is an SEM showing the resulting particle morphology
and uniform size. The diameter of the particles is estimated to be
0.54 .mu.m from FIG. 4.
Example 5
Synthesis of Polymer Particles from Low Molecular Weight Seed
Particles
[0337] The initiator emulsion and the monomer emulsion composition
were prepared as described in Example 4. In a 0.25 L reactor, 40.7
g of the seed particle dispersion prepared according to Example 2
was gently mixed with 49.9 g of the initiator emulsion. The mixture
was stirred at 25.degree. C. for 24 hours. In a 0.5 L reactor 36.3
gram of the activated seed particles and 335.0 gram of the monomer
emulsion were added. The mixture was stirred for 2 hours at
25.degree. C., then 133.4 g water was added, and then the mixture
was heated to 60.degree. C. After 1 hour at 60.degree. C. the
temperature was raised to 70.degree. C. and maintained at this
temperature for 20 hours.
[0338] The dispersion was repeatedly washed with methanol and air
dried over night followed by a further 15 hours of drying at
50.degree. C.
[0339] FIG. 5 is an SEM showing the resulting particle morphology
and uniform size. The diameter of the particles is estimated to be
0.52 .mu.m from FIG. 5.
Example 6
Synthesis of Polymer Particles from Low Molecular Weight Seed
Particles
[0340] The initiator emulsion and the monomer emulsion composition
were prepared as described in Example 4. In a 0.25 L reactor, 50.6
g of the seed particle dispersion prepared according to Example 3
was gently mixed with 47.8 g of the initiator emulsion. The mixture
was stirred at 25.degree. C. for 24 hours. In a 0.5 L reactor, 31.7
gram of the activated seed particles and 333.8 gram of the monomer
emulsion was added. The mixture was stirred for 2 hours at
25.degree. C., then 133.0 g water was added, and the mixture was
then heated to 60.degree. C. After 1 hour at 60.degree. C. the
temperature was raised to 70.degree. C. and maintained at this
temperature for 20 hours.
[0341] FIG. 6 is an SEM showing the resulting particle morphology
and uniform size.
Example 7
Analysis of Seed Particles Made Under Different Conditions
[0342] Table 1 and FIG. 7 illustrate how the size of the starting
polystyrene seed particle can be controlled by adjusting the
concentration of the surfactant SDS in the aqueous dispersion used
for emulsion polymerisation, while keeping the concentration of the
polymer and initiator constant. Diamond data points are for
syntheses including the chain transfer agent (T) and square data
points are for reactions without T. Particle sizes obtained were
from 0.058 .mu.m-0.20 .mu.m, with most from 0.09 nm to 0.16 nm. As
can be seen from the data, seed particle size is related to SDS
concentration, with a higher concentration of SDS resulting in a
smaller seed particle. The presence or absence of T during seed
synthesis did not have a significant effect on seed particle
size.
TABLE-US-00004 TABLE 1 SDS concentration and particle diameter SDS
particle polymer weight sample concentration diameter (z average
molecular ID gram/liter water average) nm weight LI541 a-1 1 177
8.4E+04 LI542 a-2 1.25 134 9.1E+04 LI532 a-3 1.5 94 1.2E+05 LI534
a-4 1.5 94 1.5E+05 LI537 a-5 1.5 95 1.4E+05 LI538 a-6 1.5 96
1.4E+05 LI509 a-7 2 58 2.4E+05 LI662 b-1 1.25 151 1.6E+04 LI728 b-2
1.25 151 1.0E+04 LI740 b-3 1.25 151 1.2E+04 LI794 b-4 1.3 144
1.2E+04 LI804 b-5 1.35 129 1.3E+04 LI670 b-6 1.6 78 7.4E+04 Samples
a-1 to a-7 are without T and b-1 to b 6 are with T. Styrene
concentration = [M] = 47 g/L water (0.45 mol/L water) Initiator
concentration = [I] = 1.7 g/L(7.5 mmol/l) ammonium persulfate. T
concentration (b-1 to b-6) = [T] = 15 mmol/L water
[0343] The presence or absence of T did, however, have a
significant effect on the molecular weight distribution of the
starting seed particles, as is apparent from Table 1. The effect of
T on molecular weight is also illustrated in FIG. 8. The molecular
weight distributions illustrated in FIG. 8 were determined with GPC
calibrated with polystyrene molecular weight standards. The curves
in the graph show the differences between 4 classes of polymer seed
particles, as follows:
[0344] 1) Trace 20: seed particle with high molecular weight.
[0345] 2) Traces 21 to 28: seed particles with a reduced molecular
weight which is controlled by reducing the amount of monomer phase
present during the seed synthesis, i.e. lowering of the particle
number i.e. surface area gives a reduced molecular weight of the
polystyrene.
[0346] 3) Traces 30 to 32: seed particles synthesised with chain
transfer agent added a short time after particle nucleation visible
appearance (i.e., the chain transfer agent was added shortly after
the solution appeared cloudy). The entire amount of chain transfer
agent is added at the same time. The molecular weight is further
reduced, but demonstrates a bimodal distribution.
[0347] 4) Traces 40 to 42: seed particles synthesised with the
chain transfer agent added over time with the initial addition
shortly after the visible appearance of particle nucleation with
visible appearance determined by the solution becoming cloudy.
[0348] As can be observed, T reduces the overall molecular weight
distribution of the polymer. It is believed that it is able to do
this by extraction of the radicals from growing polymer chains.
[0349] It is important to add T at the correct time, to obtain
monosized seed particles. The chain transfer agent should therefore
be added a short time after the particle nucleation step, either
all at once or over a longer period of time. If T is added before
particle nucleation the presence of T may alter the nucleation-step
and generate polydisperse seed particles.
[0350] Disc centrifuge analysis of the seed particle samples
plotted in FIG. 8, revealed coefficients of variation below <5%
for all of the seed particle populations.
Example 8
Analysis of Further Seed Particles
[0351] The average molecular weights were measured for a number of
other samples, as illustrated in Table 2, with samples listed as
seed type "high" and "med." ("medium") representing comparative
examples with polymer molecular weights above those of the seed
particles of the present invention. Seed type "low" represents seed
particles made according to methods analogous to those of Examples
1 to 3.
TABLE-US-00005 TABLE 2 Molecular weight data for seed particles
obtained by GPC analysis Batch-peak Component RT % SEC Weight No.
Weight Average Mw seed- No. mins Area Peak Mwt Average Mw Average
Mn sample type B650-1 5.96E+05 5.96E+05 high LI532-1 12.023 73.86
9.44E+04 1.19E+05 6.18E+04 8.76E+04 med. LI532-2 17.132 26.14
3.21E+02 2.50E+02 1.09E+02 LI541-1 12.756 99.05 4.44E+04 8.41E+04
2.93E+04 8.33E+04 med. LI541-2 18.614 0.96 3.08E+01 2.93E+01
2.77E+01 LI728-1 15.229 87.44 3.59E+03 1.01E+04 1.50E+03 8.85E+03
low LI728-2 18.719 12.56 2.85E+01 4.74E+01 3.29E+01 LI804-1 13.051
14.175 4.18E+04 6.78E+04 5.09E+04 1.28E+04 low LI804-2 15.3 55.893
4.27E+03 5.61E+03 2.07E+03 LI804-3 17.673 29.933 2.31E+02 2.07E+02
1.59E+02 LI662-1 13.224 53.13 2.78E+04 2.91E+04 1.21E+04 1.59E+04
low LI662-2 16.865 46.87 4.61E+02 8.91E+02 4.34E+02 LI735-1 12.843
35.442 4.37E+04 4.74E+04 2.42E+04 1.80E+04 low LI735-2 16.095
50.684 1.38E+03 2.26E+03 1.05E+03 LI735-3 18.635 13.874 3.56E+01
5.08E+01 3.39E+01 Note: "RT" = retention time "SEC" = size
exclusion chromatography
[0352] FIGS. 3 and 9 illustrate the CPS Disc Centrifugation results
obtained for two representative seed particle samples. The CPS Disc
Centrifugation runs used to generate the data for FIGS. 3 and 9
used a standard diameter of 0.478 .mu.m and a speed of 19500. 2969
data points were recorded for FIG. 3 over a runtime of 110.47
minutes, while 2729 data points were recorded for FIG. 9 over a
runtime of 50.74 minutes. The mode particle diameter illustrated in
FIG. 3 is 0.119 .mu.m, with a peak half width of 0.005. The mode
particle diameter illustrated in FIG. 9 is 0.186 .mu.m, with a peak
half width of 0.006. The particle diameter is an overestimate,
since the particles have a higher density than the polystyrene
standard used. Notwithstanding this, the CV % is less than 5% for
these samples.
Example 9
Analysis of Porous Polymer Particles
[0353] FIGS. 10A to 10D illustrate various porous polymer particles
obtained after expansion of seed particles according to the
Ugelstad processes. FIG. 10A illustrates, for comparison, irregular
shaped particles with diameter of approximately 0.65 .mu.m obtained
from high molecular weight seed particles. FIG. 10B illustrates,
for comparison, particles obtained from medium molecular weight
seed particles. The particles in FIG. 10B have a diameter of
approximately 0.5 .mu.m, but a broad distribution with a large CV.
The particles in FIGS. 10C and 10D illustrate particles made
according to the method of Examples 4 and 5 respectively, with
average particle diameters of 0.54 and 0.53 .mu.m. In both 10C and
10D, the observed submicron particles show a narrow distribution of
diameters, have smooth outer surfaces, and are approximately
spherical.
[0354] The CV % for size for representative samples is illustrated
in FIGS. 11 and 12. The CPS Disc Centrifugation runs used to
generate the data for FIGS. 11 and 12 used a standard diameter of
0.478 .mu.m and a speed of 19500. 1559 data points were recorded
for FIG. 11 over a runtime of 4.17 minutes, while 2472 data points
were recorded for FIG. 12 over a runtime of 67.87 minutes. The mode
particle diameter illustrated in FIG. 11 is 0.424 .mu.m, with a
peak half width of 0.047 .mu.m. The mode particle diameter
illustrated in FIG. 12 is 0.520 .mu.m, with a peak half width of
0.021 .mu.m.
[0355] The surface area of the samples has been measured with a
TriStar Surface Analyser and Porosity Analyser. For the porous
submicron polymer particles the specific surface area for particles
with 70 volume % porogen has been determined to be between 450-550
m.sup.2/g. If the particles were non porous, that is no porogen was
used in the Ugelstad process, polystyrene particles with a diameter
of 300-900 nm would be expected to have a specific surface area of
from 6 to 20 m.sup.2/g.
Example 10
Comparison of Size and Morphology of Porous Polymer Particles to
MyOne.TM. Particles
[0356] FIG. 13 is an SEM image taken at a magnification of 10,000
that provide a further comparison between porous polymer particles
made according to the invention (e.g. as illustrated by FIG. 5) and
uncoated micron sized MyOne.TM. polymer particles made according to
example 2 of WO 2005/015216 (FIG. 13). A comparison of the SEM
images of FIGS. 5 and 13 reveals important features: [0357] The
polymer particles of the present invention are smaller, that is
submicron as opposed to micron sized. [0358] The polymer particles
of the present invention have a substantially smooth outer surface
under a magnification of 10,000 as opposed to the rough and knobbly
appearance of the outer surface of the MyOne.TM. polymer
particles.
Example 11
Size and Morphology of Various Commercially Available Particles
[0359] The size and morphology of 13 commercially available
particles were obtained for comparative purposes. Particle sizes
were measured with a Coulter Counter Multisizer III.TM. according
to the standard methods described in the manufacturer's
instructions manual. The appearance was determined by examination
by light microscopy and from SEM and transmission electron
microscopy (TEM) images. The results are presented in table 3 and
FIGS. 14 to 28 provide representative SEM and TEM images.
[0360] The results demonstrate that commercially available samples
1 to 11 comprise particles that are not monodisperse, e.g. CV % is
greater than 10%. Samples 12 and 13 are monodisperse (CV<2%),
but with a measured diameter of 2.85 .mu.m. Many of these samples
also appear rough and knobbly at the magnification levels displayed
in the SEM and TEM images, e.g. in FIGS. 14 to 16, 18 to 22 and
24.
TABLE-US-00006 TABLE 3 Summary of size and morphology for
commercially available particles of approximately 1 .mu.m or larger
Stated Measured CV % (size No. Product Name Manufacturer diam.
.mu.m diam, .mu.m variation, .mu.m) Appearance 1 SeraMag SA-1, Low
streptavidin Seradyn 1.0 0.93 21.7% (0.5-1.6) Round, some small
fragments. 2 SeraMag SA-3, Medium Seradyn 1.0 0.97 22.6% (0.5-1.4)
Round, some small streptavidin fragments. 3 MagPrep Merck 1.0 0.67
37.5% (0.5-2.6) Deformed fragments of many sizes 4 ProActive
Microspheres Magnetic- Bangs 2.23 2.44 31.1% (0.5-3.6) Round, some
small Cl. Uniform 2.23 fragments many sizes 5 ProActive
Microspheres Magnetic- Bangs 0.86 0.63 40.6% (0.5-2.8) Round +
deformed Encapsulated 0.86 fragments, many sizes 6 ProActive
Microspheres Magnetic- Bangs 0.83 0.63 26.9% (0.5-1.7) Round +
deformed Classical 0.83 fragments, many sizes 7 Magnesphere Promega
0.5-1.5 0.86 43.3% (0.5-3.4) Flakes of varying size 8 BioMag
Streptavidin Ultraload Polysciences 1.0 0.67 32.8% (0.5-1.9) Flakes
of varying size 9 BioMag Streptavidin Nuclease Free Polysciences
1.0 0.67 32.4% (0.5-1.9) Flakes of varying size 10 MagPrep Novagen
1.0 0.86/0.6 36.4% (0.5-2.6) Mainly deformed, crushed particles 11
GenoPrep GenoVision/Qiagen unknown 0.86 19.6% (0.5-1.6) Round, some
small fragments. 12 Dynabeads M270-Streptavidin Dynal Biotech ASA
2.8 2.85 1.24% Monodisperse and monosized particles 13 Dynabeads
M280-Streptavidin Dynal Biotech ASA 2.8 2.72 1.26% Monodisperse and
monosized particles
Example 12
Nitration of Porous Polymer Particles
[0361] Porous polymer particles with a diameter of 0.45 .mu.m were
made in accordance with examples 4-6 from a low molecular weight
seed particle of the disclosure.
[0362] In a 2 l glass vessel there was added 1240 gram concentrated
sulfuric acid. The reactor was equipped with a jacketed glass
reactor for temperature regulation and a teflon stirrer.
[0363] The acid was cooled down to 7.degree. C. and 377 g nitric
acid (65%) was added over a 15 minute interval, the reaction
mixture reaching a maximum temperature of 28.degree. C. The acid
solution was cooled down to 6.degree. C. 50 gram of dry porous
polymer particles were added and the dispersion was heated to
30.degree. C. for 90 minutes. The dispersion was then poured into 5
kg of ice-water, followed by purification of the nitrated polymer
particles with water and methanol.
[0364] FTIR analysis showed strong adsorption at 1531 cm.sup.-1
confirming the aromatic substitution of nitro groups. The reaction
yield was 61.5 gram of dry nitrated particles.
Example 13
Magnetisation of Nitrated Porous Polymer Particles
[0365] 300 gram dispersion of nitrated particles (4.8 weight % in
water) from example 12 was added to a 0.5 litre jacketed glass
reactor. 83 g of iron(II) sulfate heptahydrate and 0.11 g of
manganese(II) sulfate hydrate were added. The mixture was stirred
for 30 minutes to dissolve the iron salt. After 30 minutes, 116
gram 25% ammonia was added while stirring. The dispersion turned
black immediately and was further heated to 60.degree. C. and kept
at 60.degree. for 2 hours. The magnetic particles were purified
with several centrifugal shifts to remove unbound magnetic
material.
Purification Procedure:
[0366] The dispersion was concentrated and transferred to a 1 liter
centrifugal bottle and then there was added 1 liter diluted ammonia
(0.1v % in water). The dispersion was set on a shaker to ensure a
homogeneous dispersion. Then the supernatant was separated from the
magnetic polymer particles by centrifugation and discarded. This
procedure was repeated 5 times (*1 liter) with diluted ammonia
(0.1v %) and then 10 times (*1 liter) with purified water.
[0367] The final magnetic polymer particles contained 490 mg/g iron
oxide determined by elemental analysis.
Example 14
Coating
[0368] 7.5 g of magnetic particles from example 13 dispersed in 61
g diethyleneglycol dimethylether were added a 200 mL reactor. The
reactor was placed in a temperature controlled water bath and
equipped with a stirrer.
[0369] For a pre-coating step 1.4 gram of butanediol
diglycidylether+bisphenol A diglycidylether (Araldite.RTM. LY 564)
was added and the dispersion was heated to 75.degree. C. and kept
at 75.degree. C. for 3 hours. After cooling down the solution 15.0
g butanediol diglycidylether, 6.0 g glycidol, 11.0 g
glycidolmethacrylate and 6.0 g diethylene glycoldimethylether were
added. The dispersion was heated to 75.degree. C. and kept at
75.degree. C. for 18 hours. The dispersion was purified by
separating the particles from the supernatant with an external
magnet, and washed 4 times with 100 mL methanol and 4 times with
100 mL methanol/isopropanol mixture (30/70 v %).
[0370] The measured yield of the coated magnetic particles was 8.8
g.
[0371] FTIR analysis confirmed the incorporation of epoxy coating
and vinyl functional groups.
Example 15
Polyacrylic Acid Modified Beads
[0372] 6.5 g of coated magnetic particles from example 14 were
dispersed in 24.5 gram methanol/isopropanol mixture (30/70 v %) and
charged in 100 mL reactor. The reactor was equipped with a stirrer,
a cooler and placed in an oil bath with temperature regulation.
[0373] In a separate 50 mL beaker 0.4 gram azobisisobutyronitrile
(AIBN) was dissolved in 19.9 mL methanol/isopropanol mixture
(30/70v %).
[0374] The dissolved azobisisobutyronitrile and 7.8 gram acrylic
acid were added to the particle dispersion, heated to 75.degree. C.
and kept at 75.degree. C. for 19 hours.
[0375] The dispersion was purified by separating the particles from
the supernatant with an external magnet, and washed 6 times with 40
mL methanol and finally dispersed in 60 mL 0.15 M sodium hydroxide
solution. The dispersion was heated to 75.degree. C. for 4 hours.
The particles were purified with water by consecutive magnetic
separations.
[0376] The final particle had a mode diameter of 0.5 .mu.m with a
narrow size distribution determined by disc-centrifugation. FTIR
confirmed the incorporation of carboxylic acid groups and the acid
content was determined to be 0.8 mmol/g dry weight by
titration.
Example 16
Compact Beads with High Cross Linking
[0377] 15.5 g SDS, 1290 g water, 0.1 g Synperonic.TM. A11 (a
polyoxyethylene (11) C12-C15 alcohol emulsifier), 15.5 g acetone
and 129 g dioctanoylperoxide were mixed with an Ultra-Turrax
disperser and homogenised with a pressure homogeniser (=initiator
emulsion)
[0378] 503.6 g of a low molecular weight seed dispersion (4.57 w %
dry content) made according to example 1 and with a measured seed
diameter of 0.11 .mu.m was gently mixed with 194.3 g of the
initiator emulsion. The mixture was stirred at 25.degree. C. for 24
hours (=activated seed particles).
[0379] 30.6 g styrene, 37.0 g glycidylmethacrylate, 117.4 g
divinylbenzene (comprising 65 g divinylbenzene and 45 g
ethylvinylbenzene), 307 g water and 1.3 g SDS were charged to a 1
liter reactor and 268 g of the activated seed particles were added.
After stirring for 4 hours at 25.degree. C., 238 g water was
charged and the temperature was raised to 60.degree. C. and kept at
60.degree. C. for 2 hours and further 70.degree. C. for 5
hours.
[0380] To introduce amine functional groups 15.6 g ethylene diamine
was charged to 500 ml of the particle dispersion and the mixture
was heated to 80.degree. C. and kept at 80.degree. C. for 2
hours.
[0381] The compact polymer particles have a mode diameter of 0.3
.mu.m measured by disc centrifugation.
Example 17
Silica Coated Submicron Monosized Magnetic Particles
[0382] Monosized 0.5 .mu.m magnetic particles were made according
to reaction scheme 1 and examples 12 and 13.
[0383] In a reactor a mixture of 2 g magnetic 0.5 .mu.m particles
and 6.3 g absolute ethanol was stirred at room temperature. Then
2.4 g tetraethyl orthosilicate, 98%, 24.0 g water and 8.0 mL
ammonium solution, 28%, were charged. The mixture reacted for 18
hours at room temperature. The particles were purified by magnetic
separation of the coated particles from the supernatant. The
reaction yield was 26 g and FTIR analysis confirmed the presence of
Si--O groups.
Example 18
Comparative Example Using High Molecular Weight Seed Particles
[0384] A high molecular weight seed (M.sub.w 4.6.times.10.sup.5)
with a diameter of 0.15 .mu.m was used to make two porous particle
dispersions with different pore volumes.
[0385] The synthesis procedure followed the same steps as outlined
in example 4 but with a initiator/seed (Y/P) ratio of 1 and a
monomer to polymer ratio of 35 (M/P).
[0386] SEM images showed that the resulting particles appeared
cornered and not spherical, and with a broad size distribution.
Example 19
Distribution of Magnetic Material
[0387] An SEM is made of a cross-section of a magnetic polymer
particle made by the Ugelstad process. The particle has a diameter
of 2.8 .mu.m and therefore falls outside the scope of the invention
but, nevertheless, serves to illustrate that the incorporation of
magnetic material in polymer pores does not change the particle
morphology. The SEM is shown in FIG. 28, where iron oxide (i.e.
magnetic material) shows as bright points, illustrating how the
magnetic material is dispersed throughout the interior of the
particle (in pores) without clumping and without changing the
external morphology.
* * * * *