U.S. patent application number 11/333624 was filed with the patent office on 2006-08-24 for process.
This patent application is currently assigned to DYNAL BIOTECH ASA. Invention is credited to Arvid Trygve Berge, Geir Fonnum, Lars Kilaas, Grete Irene Modahl, Tom-Nils Nilsen, Ruth Schmid.
Application Number | 20060188905 11/333624 |
Document ID | / |
Family ID | 36913179 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188905 |
Kind Code |
A1 |
Fonnum; Geir ; et
al. |
August 24, 2006 |
Process
Abstract
A process for the preparation of coated polymer particles
containing superparamagnetic crystals, said process comprising
reacting surface-functionalized, superparamagnetic
crystal-containing polymer particles of diameter less than 0.5
.mu.m with at least one polyisocyanate and at least one diol.
Inventors: |
Fonnum; Geir; (Fjellhamar,
NO) ; Kilaas; Lars; (Skansegt, NO) ; Berge;
Arvid Trygve; (Falsensgt, NO) ; Nilsen; Tom-Nils;
(Fjordglottv, NO) ; Schmid; Ruth; (Tiller, NO)
; Modahl; Grete Irene; (Oslo, NO) |
Correspondence
Address: |
Paul D. Greeley, Esq.;Ohlandt, Greeley, Ruggiero & Perle, L.L.P.
10th Floor
One Landmark Square
Stamford
CT
06901-2682
US
|
Assignee: |
DYNAL BIOTECH ASA
|
Family ID: |
36913179 |
Appl. No.: |
11/333624 |
Filed: |
January 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60658736 |
Mar 4, 2005 |
|
|
|
Current U.S.
Class: |
435/6.15 ;
427/2.11; 435/7.5; 436/526 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
2563/143 20130101; C12Q 2563/149 20130101; C12Q 1/68 20130101; G01N
33/5434 20130101; Y10T 428/2998 20150115; Y10T 436/143333
20150115 |
Class at
Publication: |
435/006 ;
435/007.5; 436/526; 427/002.11 |
International
Class: |
G01N 1/28 20060101
G01N001/28; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53; G01N 33/553 20060101 G01N033/553 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2005 |
GB |
0500888.3 |
Claims
1. A process for the preparation of coated polymer particles
containing superparamagnetic crystals, said process comprising
reacting surface-functionalized, superparamagnetic
crystal-containing polymer particles of diameter less than 0.5
.mu.m with at least one polyisocyanate and at least one diol.
2. A process as claimed in claim 1 wherein at least two diols are
employed.
3. A process as claimed in claim 1 wherein said diol is selected
from the group consisting of polyethylene glycols and diols of
formula HO((CH.sub.2).sub.mO).sub.nH (where n is an integer of 1 to
15 and m is an integer of 2 to 6).
4. A process as claimed in claim 2 wherein said diols are
diethyleneglycol and tetraethyleneglycol.
5. A process as claimed in claim 2 wherein one of said diols is
polyethyleneglycol.
6. A process as claimed in claim 1 wherein said polyisocyanate is
4,4-methylene bis(phenylisocyanate) or a polyisocyanate comprising
4,4-methylene bis(phenylisocyanate) with CH.sub.2-phenylisocyanate
residues (Desmodur.TM.).
7. A process as claimed in claim 1 wherein said polyisocyanate is a
diisocyanate.
8. A process as claimed in claim 1 wherein said particles are, in a
first stage, reacted with a mixture of polyisocyanate and at least
one diol in which the polyisocyanate is in a molar excess relative
to the diol(s) and, in a second stage, subsequently reacted with at
least one diol.
9. A process as claimed in claim 8 wherein two diols are used in
the first stage of the reaction and one or two diols used in the
second stage.
10. A process as claimed in claim 9 wherein diethyleneglycol and
tetraethyleneglycol are used in both stages of the reaction.
11. A process as claimed in claim 1 wherein the particles are, in a
first stage, reacted with polyisocyanate in the absence of diol,
and, in a second stage, reacted with at least one diol.
12. A process as claimed in claim 11 wherein one dial is employed
and said diol is a polyethylene glycol.
13. A process as claimed in claim 1 wherein the particles are amine
functionalised.
14. A process as claimed in claim 1 wherein said
surface-functionalized polymer particles are nitrated and reduced
styrene-divinylbenzene polymer particles.
15. A process as claimed in claim 1 wherein the diameter of the
polymer particles is between 150 and 250 nm.
16. A process as claimed in claim 1 in which said coated particle
is subsequently tosylated.
17. A process as claimed in claim 1 wherein subsequent to the
coating reaction, said particles are coupled to a drug molecule,
reporter moiety or ligand.
18. A process as claimed in claim 17 wherein said ligand is a
monoclonal antibody, polyclonal antibody, antibody fragment,
nucleic acid, oligonucleotide, protein, oligopeptide,
polysaccharide, sugar, peptide, peptide encoding nucleic acid
molecule, antigen or drug.
19. A process as claimed in claim 18 wherein said ligand is
streptavidin.
20. A particle obtainable by the process of claim 1.
21. A method of using the particle as claimed in claim 20 in an
assay.
22. A method as claimed in claim 21 wherein said assay is for the
detection of nucleic acid or is an immunoassay.
23. A method of using the particle as claimed in claim 21 in
hydrophobic interaction chromatography or reversed phase
chromatography.
Description
CROSS REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This instant patent application claims priority to British
Patent Application No. GB0500888.3 filed on Jan. 17, 2005, and U.S.
Provisional Patent Application Ser. No. 60/658,736 filed on Mar. 4,
2005, which are herein incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to a process for the preparation of
coated magnetic polymer particles.
[0004] 2. Description of the Related Art
[0005] Magnetic polymer particles are of general utility in various
medical and biochemical fields, for example as transport vehicles
for the delivery of pharmaceutical products, for diagnostic
purposes, for separation and for synthetic purposes. Such particles
rely upon their magnetic properties in order to perform these
functions. In diagnostic assay applications, for example,
application of a magnetic field to a sample containing an analyte
bound to magnetic polymer particles allows the isolation of the
analyte without the use of centrifugation or filtration and in
therapeutic applications, for example, application of a magnetic
field to the patient may serve to target drug-carrying magnetic
polymer particles to a desired body site.
[0006] By magnetic is meant herein that the polymer particles
contain superparamagnetic crystals. Thus the magnetic polymer
particles are magnetically displaceable but are not permanently
magnetizable. Many processes for preparing magnetic polymer
particles are known, a large number of which involve preparing
maghemite- or magnetite-containing polymer particles from
pre-formed magnetic iron oxides, e.g. magnetite. Some of processes
involved are described in U.S. Pat. No. 4,654,267 (Ugelstad) the
contents of which are incorporated herein by reference.
[0007] With the development of devices which can measure very small
changes in magnetism as well as the growth in nanotechnology it is
envisaged that magnetic particle users will desire smaller
particles with which to conduct their assays etc in the future.
Moreover, the magnetic particles will need to be manipulated to
carry reactive groups which can be readily coupled with labels to
provide an ideal solid phase for biochemical procedures.
DETAILED DESCRIPTION OF THE INVENTION
[0008] We have now surprisingly discovered that magnetic particles
with particularly useful surface characteristics may be produced by
reacting surface functionalized, magnetic polymer particles with a
combination of polyisocyanate/diol monomers to produce a "coated"
magnetic polymer particle. Such particles are readily coupled
further with labels to provide a solid phase support useful in a
variety of fields.
[0009] Viewed from a first aspect, therefore, the invention
provides a process for the preparation of coated polymer particles
containing superparamagnetic crystals, said process comprising
reacting surface-functionalized, superparamagnetic crystal
containing polymer particles of diameter less than 0.5 .mu.m with
at least one polyisocyanate, e.g. diisocyanate, and at least one,
preferably at least two, diols.
[0010] Preferred diols are polyethylene glycols or are of formula
HO((CH.sub.2).sub.mO).sub.nH (where n is an integer of 1 to 15,
e.g. 2 to 10, preferably 2 to 4, and m is an integer of 2 to 6,
preferably 2 to 3, most preferably 2). Where only one diol is
employed, this is preferably a polyethylene glycol, e.g.
polyethylene glycol 300, 400, 500 or 600.
[0011] The polymer particles used in the process of the invention
may be any suitably sized, optionally porous polymer particles
having a functionalized surface, e.g. Ugelstad particles as
generally described in U.S. Pat. No. 4,654,267. Suitable particles
are also available from Ademtech, e.g. Carboxyl-Ademtech 0212
particles which comprise a carboxylic acid surface coating.
[0012] The surface functionality on the polymer particle is
preferably a group capable, optionally with activation, of reacting
with a polyisocyanate to covalently bond the polyisocyanate to the
surface. Most preferably the surface is amine or carboxyl
functionalized.
[0013] The polymer particle is preferably made from combinations of
vinylic polymers, e.g. styrenes, acrylates and/or methacrylates.
The polymeric material may optionally be crosslinked, for example
by incorporation of cross-linking agents, for example as
comonomers, e.g. divinylbenzene (DVB) or ethyleneglycol
dimethacrylate. Particles comprising DVB are preferred.
[0014] Appropriate quantities of the cross-linking agents (e.g.
comonomers) required will be well known to the skilled man.
Preferably the polymer is a cross-linked styrenic polymer (e.g. a
styrene-divinylbenzene polymer, which may be surface functionalized
by the use of a nitro-group containing comonomer, e.g.
nitro-styrene, and subsequent reduction) or a cross-linked
(meth)acrylic polymer surface functionalized by the use of an
epoxy-group containing comonomer (e.g. glycidylmethacrylate) and
subsequent amination (e.g. by reaction with ethylene diamine).
[0015] The superparamagnetic crystals in the polymer particles used
in the process of the invention 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. Magnetic iron oxides, e.g. magnetite or maghemite are
preferred; however the crystals may be of mixed metal oxides or
other magnetic material if desired. The total quantity of
crystalline magnetic material present is generally more than 20%,
preferably more than 25%, desirably more than or equal to 30% (by
weight, e.g. up to 85% wt or at least 50 wt %, e.g. 30 to 80 wt %.
The percentage is calculated on a Fe (or equivalent metal in the
case of magnetic materials other than iron oxides) weight basis
based upon the overall dry weight of the coated particles.
[0016] Higher concentrations of magnetic crystals in the polymer
particles are required for the small particles claimed herein so
that they can be successfully attracted by a magnet during any
isolation procedure. Another way of increasing the ease that beads
are dragged towards a magnet is to make a portion of them
remanent.
[0017] The degree of remanency may be measured by a vibrating
sample magnetometer by running hysteresis curves. The curves are
produced by measuring the magnetisation of the particles at
different field strengths going from, for example, -1.5 tesla to
+1.5 tesla and back. If the maximum magnetisation is called Ms and
the absolute value of crossing the Y-axis (Zero Field strength) is
called Mr, the ratio Mr/Ms will tell how remanent the particles
are.
[0018] Particles with a ratio of Mr/Ms lower than 0.15 can for most
application be considered superparamagnetic. The time interval for
going from -1.5 to +1.5 tesla should be 10 minutes. Particles with
low values of remanency often have increased drag towards a magnet
because of aggregation, but the forces between them are so small
that this do not influence sample washing.
[0019] Porous polymer particles may have magnetic particles
deposited in their pores by standard techniques. As a further
possibility, porous polymer particles may be prepared from nitro
styrene and DVB, and magnetic material introduced. The use of amino
styrene, particularly 4-aminostyrene, as monomer or comonomer in
the preparation of amino-bearing polymeric material is preferred.
Use of this monomer or comonomer obviates the need for
post-polymerisation nitration and reduction reactions. Moreover,
the more predictable nature (homogeneity) of the coating afforded
by this process permits a more reliable coating to be applied.
[0020] The polymer particles may be formed carrying surface
functionalisation or alternatively, 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 amino ethanol.
[0021] Polymer particles according to the present invention will
have sizes (i.e. diameters) that are less than 500 nm, e.g.
generally in the range from 100 nm to 400 nm, e.g. 150 nm to 250
nm.
[0022] Typically the particles used will have a surface area of at
least 15 m5/g (measured by the BET nitrogen absorption method), and
more preferably at least 30 m5/g, e.g. up to 700 m5/g, when
corrected to a mean particle diameter of 2.7 .mu.m (i.e. multiply
surface area by 2.7/MD, where MD is the mean diameter in
micrometers). Similarly scaled, the particle pore volume is
preferably at least 0.1 mL/g.
[0023] Typically, the polymer particles are spherical and
substantially monodisperse before they are coated and especially
preferably remain spherical and substantially monodisperse once
they have been coated.
[0024] By substantially monodisperse it is meant that for a
plurality of particles (e.g. at least 100, more preferably at least
1000, e.g. all) the particles have a coefficient of variation (CV)
of less than 20%, for example less than 15%, preferably less than
12%, more preferably less than 11%, still more preferably less than
10% and most preferably no more than about 8%, e.g. 2 to 5%. CV is
determined in percentage as CV = 100 .times. standard .times.
.times. deviation mean ##EQU1## where mean is the mean particle
diameter and standard deviation is the standard deviation in
particle size. CV is preferably calculated on the main mode, i.e.
by fitting a monomodal distribution curve to the detected particle
size distribution. 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 Coulter LS 130
particle size analyzer.
[0025] The reaction of the magnetic polymer particle with the
polyisocyanates generates a polymer coating. When the particles are
porous, the resulting "coated" particles then have reduced porosity
relative to the porous starting material. Surprisingly we have
found that the superparamagnetic crystals appear to catalyse the
polymerization so that the coating forms preferentially in their
vicinity.
[0026] In another preferred embodiment, the coating polymer is
formed from one or more (e.g. 1, 2 or 3) polyisocyanates and one or
more (e.g. 2, 3 or 4) diols. Preferably, one polyisocyanate should
be employed, e.g. one diisocyanate. Alternatively, a mixture of
closely related polyisocyanates can be employed (e.g.
Desmodur).
[0027] Typical polyisocyanates which may be used include methylene
diisocyanate, hexamethylene diisocyanate, 2,4-toluene diisocyanate
(2,4-TDI) (and isomers or mixtures thereof), isophorone
diisocyanate (IPDI), 4,4'-oxybis(phenylisocyanate),
4,4'-diphenylmethane diisocyanate (MDI), mixtures of MDI and
oligomers based on MDI (e.g. Desmodur VL),
2,4-diphenyldiisocyanate, methylene biscyclohexyl diisocyanate
(H.sub.12MDI), phenylene diisocyanate (p-PDI),
trans-cyclohexane-1,4-diisocyanate (CHDI), 1,6-diisocyanatohexane
(DICH), 1,5-diisocyanato-naphthalene (NDI), paratetramethylxylene
diisocyanate (p-TMXDI) or metatetramethylxylene diisocyanate
(m-TMXDI).
[0028] An especially preferred isocyanate is MDI or polyisocyanates
based thereon (e.g. Desmodur). Desmodur comprises MDI and oligomers
thereof comprising MDI with CH.sub.2-phenylisocyanate residues. The
Desmodur is thus a mixture of various polyisocyanates deriving from
MDI. A sample structure may be [0029] 4,4-methylene
bis(phenylisocyanate) 40-50% [0030] 4,4-methylene
bis(phenylisocyanate)+benzylisocyanate: 20-25% [0031] 4,4-methylene
bis(phenylisocyanate)+2 benzylisocyanate: 10% [0032] 4,4-methylene
bis(phenylisocyanate)+3 benzylisocyanate: 2%.
[0033] (In a reaction like this the product also contains some of
the 2-isomer). The compound is sold by Shell under the trade name
Caradate and under the trade names Hylene and Rubinate by
Huntsman.
[0034] Preferably two diols should be employed. The diols are
preferably used in a molar ratio of 0.5:1 to 1:0.5, more preferably
0.8:1 to 1:0.8 when two diols are used. Preferably no one diol is
used in a quantity exceeding 90% mol. of the diol mixture.
[0035] Preferred diols include diethylene glycol, tetraethylene
glycol and polyethylene glycols e.g. PEG 300, 400 or 600. A
preferred diol combination is diethylene glycol and tetraethylene
glycol.
[0036] During the coating reaction involving the polyisocyanate, it
is preferred if, in a first stage the polyisocyanate is in excess
(e.g. relative to any diol). It is within the scope of the
invention to use only polyisocyanate in this step of the coating
procedure. This is believed to minimise the possibility of gelling
occurring during the reaction. Where a large excess of
polyisocyanate is employed in an initial coating reaction, it may
then be necessary to react, in a second stage, the coated particles
with further diol(s) (e.g. a diol as described above) to react with
any unreacted isocyanate groups. Where the initial coating reaction
uses polyisocyanate alone, it is essential that the resulting
particle is reacted with at least one diol thereafter.
[0037] In such an embodiment, such a diol is preferably a
polyethylene glycol. The long chain of the PEG diol allows the
formation of a sizable linker between the particle coating surface
and hence makes easier reaction with affinity ligands such as
streptavidin.
[0038] It is thus within the scope of the invention to react the
particles with polyisocyanate followed by diol, i.e. a stepwise
process, to effect coating.
[0039] Typically therefore, the coating reaction may be effected by
impregnating the porous magnetic polymer particle with the
polyisocyanate and diol(s), e.g. using a solution of these (for
example in an organic solvent such as methanol or diglyme) or by
mixing a dispersion of the particles in an organic solvent with a
liquid diol/polyisocyanate mixture. Sonication may be used to
improve impregnation and the reaction may be accelerated by raising
the temperature, e.g. to 50-100.degree. C. Any solvent used may be
extracted by application of sub-ambient pressure.
[0040] Generally, the uses to which magnetic polymer particles are
put, e.g. their use as diagnostic tools, require an appropriate
degree of electrophilicity in order that they may participate
adequately in coupling and other reactions in aqueous systems
prevalent in biological media.
[0041] Whilst the general polarity of the coatings is desirably
electrophilic, certain coatings which contain hydrophobic moieties
may be incorporated so as to tailor the degree of electrophilicity
to that which is desired. In this way, the invention permits the
provision of useful diagnostic and other tools having a wide range
of polarities.
[0042] If desired the surfaces of the coated magnetic polymer
particles may be further functionalised, 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).
[0043] 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 magnetic polymer particles are to be used for the
targeted release of an active compound. Accordingly after coating
has been effected, the pendent groups of the coating may be
manipulated to provide appropriate functionality (for example
epoxy, hydroxy, amino etc. functionalities) for the attachment of
such substances.
[0044] 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.
[0045] Viewed from a further aspect the invention provides the use
of particles of the invention in syntheses, extractions or assays,
in particular in nucleic acid detection.
[0046] Introduction of vinyl groups polymerisable with, for
example, an acrylic acid can also be achieved by reacting the
coating surface with a compound such as methacrylic anhydride. For
example, a coated particle which has reacted with a diol might
carry hydroxyl functionalities which would react readily with
methyl acrylic anhydride to allow the introduction of vinyl groups
to the polymer surface.
[0047] As mentioned above, the nature of the external substance
coupled to the particles may be selected on the basis of its
ability to bind to a particular target material. Nucleic acid
detection generally involves probing a sample thought to contain
target nucleic acids using a nucleic acid probe that contains a
nucleic acid sequence that specifically recognises, e.g. hybridises
with, the sequence of the target nucleic acids, such that the
nucleic acid affinity ligand and the target nucleic acids in
combination create a hybridisation layer. Suitably functionalised
particles of the invention, e.g. carrying streptavidin, are ideally
suited for nucleic acid detection.
[0048] Biotinylated single strand oligonucleotide probes bound to
streptavidin particles can be used to isolate sequence specific
DNA. The biotinylated probes are bound to the particles by mixing
the appropriate amount of particles with an excess of biotinylated
probe. The particles/probe are then incubated with the DNA sample
in a hybridisation buffer, e.g. SSPE or SSC, under conditions
appropriate for the length and sequence of the probe and DNA. The
excess and unwanted DNA is washed away utilizing the magnetic
properties of the particles. The captured DNA can be
detected/quantified by PCR etc.
[0049] Biotinylated double strand DNA fragments bound to
streptavidin particles can be used to isolate DNA sequence specific
binding proteins. The biotinylated DNA is bound to the particles by
mixing the appropriate amount of particles with an excess of
biotinylated DNA fragments. The particles/DNA are then incubated
with the protein sample in a hybridisation buffer, under conditions
appropriate for the protein under investigation. The excess and
unwanted protein is washed away utilizing the magnetic properties
of the particles. The captured protein can be eluted from the probe
(by high salt, low salt, heat, low pH etc) for downstream
applications and detection.
[0050] The target material may optionally be a material of
biological or synthetic origin, e.g. it may be a molecule or a
group of molecules, including for example antibodies, amino acids,
proteins, peptides, polypeptides, enzymes, enzyme substrates,
hormones, lymphokines, metabolites, antigens, haptens, lectins,
avidin, streptavidin, toxins, poisons, environmental pollutants,
carbohydrates, oligosaccharides, polysaccharides, glycoproteins,
glycolipids, nucleotides, oligonucleotides, nucleic acids and
derivatised nucleic acids, DNA, RNA, natural or synthetic drugs,
receptors, virus particles, bacterial particles virus components,
cells, cellular components, natural or synthetic lipid vesicles,
polymer membranes, polymer services and particles and glass and
plastic surfaces.
[0051] Where the particles of the invention are to be employed in
immunoassays it has surprisingly been found that tosylation of the
particles after coating results in particles which exhibit improved
performance in immunoassays. Thus, in a preferred embodiment,
particles carrying a coating can be tosylated, e.g. by reaction of
the particles with tosylchloride in the presence of a base. The
resulting tosylated coated particles are new and form a further
aspect of the invention. By tosyl is meant a toluene-4-sulphonyl
group.
[0052] Moreover, such tosylated species can be readily reacted with
affinity ligands, e.g. streptavidin to form still further new
particles.
[0053] Thus viewed from a further aspect, the invention provides
coated polymeric particles, carrying superparamagnetic crystals,
having a coating formed from at least one polyisocyanate and at
least one diol, which is subsequently tosylated, e.g. by reaction
with tosyl chloride and optionally then reacted with an affinity
ligand, e.g. streptavidin.
[0054] Moreover, it has surprisingly been found that particles of
the diameters claimed herein have a greatly increased capacity for
binding compared to particles of greater size, e.g. 3 .mu.m
particles. It is envisaged that the binding capacity of the claimed
particles is over 200% greater than that of larger particles
allowing the use of considerably lower amounts particles in an
assay procedure.
[0055] The particles of the invention are therefore of utility in
adsorption/desorption processes analogously to the mechanisms in
Reversed Phase chromatography or hydrophobic interaction
chromatography. Reversed phase chromatography is a separation
technique that utilises a hydrophobic adsorption interaction
between a solute molecule (e.g. a protein) and an immobilised
hydrophobic ligand (e.g. the surface of particles). This
interaction is usually so strong that it can occur in solutions of
low ionic strength and is broken by the use of organic solvents
(e.g. acetonitrile). Reversed phase chromatography can be used to
fractionate complex protein samples and for desalting protein
samples. RPC is usually performed using a solid phase packed in to
a column. The particles of the invention enable the technique to be
performed without a column, without sample dilution and to be
automated with high throughput.
[0056] Hydrophobic interaction chromatography (HIC) is a separation
technique that utilises a hydrophobic adsorption interaction
between a solute molecule (e.g. a protein) and an immobilised
hydrophobic ligand (e.g. the surface of particles). This
interaction is weaker than the interactions utilised during RPC and
requires promotion by high salt concentrations. Consequently,
decreasing salt concentrations can be used to break these
adsorption interactions. HIC can be used to fractionate complex
protein samples and for desalting protein samples. HIC is usually
performed using a solid phase packed in to a column. The particles
of the invention enable the technique to be performed without a
column, without sample dilution and to be automated with high
throughput.
[0057] The invention will now be described further by reference to
the following example.
EXAMPLE 1
Polyurethane Coating of 0.3 .mu.m Magnetic Carboxylic Acid
Particles
[0058] 6.5 gram of a magnetic particles dispersion (0212.times.
from Ademtech), with a dry content of 3.1% by weight were added to
a reaction vessel and placed on a magnet. The water phase was
removed from the particles and the particles were washed three
times with 5 mL 0.01M sodium hydroxide, twice with 5 mL 0.01M
hydrochloric acid, again with 0.01M sodium hydroxide and then with
pure water. Further the particles were washed with methanol and
transferred to diethyleneglycoldimethylether. The concentration of
particles in diethyleneglycol dimethylether were adjusted to the
original value of 3.1% by weight.
[0059] 0.5 gram of diphenylmethane diisocyanate (Desmodur vl,
Bayer) was added, and the reaction vessel were placed on a vortex
with a heating block. The temperature was set to 80.degree. C. for
20 hours. Infrared spectroscopy showed that the particles have
attached urethane groups and isocyanate groups (wave number 1707
cm.sup.-1 for C.dbd.O stretch and 2278 cm.sup.-1 for the
N.dbd.C.dbd.O stretch).
[0060] The particle dispersion from above were added 0.6 gram of
polyethylene glycol 300 and heated to 80.degree. C. for 1 hour. The
particles were washed twice with 20 mL diethyleneglycol
dimethylether and further six times with 20 mL methanol and then
transferred to water (3.times.20 mL). Infrared spectroscopy showed
that the polyethylene glycol were covalently attached to the
particles by a reduction in the N.dbd.C.dbd.O stretch at 2278
cm.sup.-1 and an increase in the ether groups at (1112 cm.sup.-1
and 1071 cm.sup.-1).
* * * * *