CA1266769A - Magnetic particles for use in separations - Google Patents

Abstract

ABSTRACT
A process is provided for the preparation of magnetic particles to which a wide variety of molecules may be coupled. The magnetic particles can be dispersed in aqueous media without rapid settling and conveniently reclaimed from media with a magnetic field. Preferred particles do not become magnetic after application of a magnetic field and can be redispersed and reused. The magnetic particles are useful in biological systems involving separations.

Description

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l. IELD OF T~E I ~ NTION

This invention relates to ~agnetically re~ponsive parti~les and to their use in 6y~tem~ i.n which the separation of certain molecules fr~m the ~urrounding medium i necessary or desirable. ~ore! particularly, the invention relat*s to ~ethods ~or the preparation of ~agnetically responsive particle~ comprising a metal oxine core surrou~ded b~ a stable ~ilane coating to ~hich a wide variety of organic ~nd/or biological ~olecules may be : coupled. The particles (coupled or uncoupled) c~n be disper~ed in aqueous media without r~pid gravitational settling and conveniently reclaimed from the media with a magnetic field. Preferably, the proce~s provided herein yields particles that are superpar~m~gnetic; that is, they do not become permanently magnetized after application of a magnetic field. Thi~ property permi~s the particles ~o be redispersed without magnetic aggregate formation.
~ence the particles may be reused or recycled. Stability of the ~ilane coating and the covalent attacbent of molecules thereto ~lso contribute to particle use an~
xeuse.
~ he magnetically respon~ive particles of this invention may b~ coupled to ~iologi~al or organic molecules with affinity ~or or the ~bility to adsorb or which interact with certain other biological or organic molecules. Particle~ ~o coupled may be used in a var iety of in vitro ~r in vivo &y~tems involving separation ~teps or the directed movement of coupled ~olecules to par~icular site~, including, bue not limited to, immunologic~l ass~ys, oth~r biological assays, bioche~ical or enzy~atic reactions, ~ffinity chro~atogr~ph~c purification~, cell ~orting and diagnostic and therapeutic uses.
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2. BACKGROUND 0~ T~E INVENT~ON
2.1. MAG~FTIC SEPARA~IONS IN ~I~LOGICAL
STE~S-_ G~NE M L CDN5lD-~A5-0:-The u~e of ~agneti~ separationl~ ~n biol~gicalsystems a~ ~n alternative to gravitational or centrifugal ~eparations ha~ be~n revie~ed lB.L~ ~irlschbein et al.~
Chemtech, March 19~2sl7~ 179 ~1982); ~. Pourfarzaneh, The Ligand Quarterly 5tl~:41-47 ~1982); ~nd POJ. ~alling and tO ~. Dunnill, En~yme Microb. ~echnol. 2s~ 10 (1980)~o 5everal advantayes of using magn~tically separable particle~ as supports for biological molecule~ 6uch as enzymes, antibodies and other bioaffinity adsorbent~ are generally recognized. ~or in~t~nce, when ~agnetic parSicles are u~ed as 601id phase 8upport& in imMobili~ed enzyme systems Isee, e.~., P.J. Robinson e~ al., Biotech.
Bioeng.~ XV:60~ 6Q6 (1973)], the enzyme may be selectively recovered from media, including media conta1ning ~u6pended ~olids, allowing recycling in enzyme re~ctor~. When used as solid ~upports in immunoassays or other competitive binding assays, magnetic particle~ permit homogene~us reaction ~onditions twhich promote optimal binding ki~etic~ and minimally alter analyt~ ads~rbent equilibrium) and facilitate separation of ~ound from unbound analyte, compared to centri~uga~ion. Centrifu~al separations are ~im~ consuming, require expensive and energ~ consumin~ equipment and po e radiological, biolvgical and physical haz~rdc. ~agnetic separations, on the other hand, are relatively ~apid ~nd e~sy, ~e~ui~ing simple equipmentO Finally, the u~e of no~ porou~
~dssÆbent-coupled ~agnetic particle~ in af~-inity chromatography ystems allows better ~ss ~ran~fe~ ~nd result~ in les. fouling than in co~ventional ~f~inity chro~atography systems.

-Although the gener~l concept of ~agnetizing molecules by coupling them to magne~ic particle6 has been discussed and the potential ~dvantage~ of usin~ Quch particles for biological purpose~ recogni~ed, the practical development of magnetic separations ha~ been hindered by several critical propertie~ of magnetic particle~ developed thus ~ar.
; Large magnetic particles ~mear~ diameter in ~olution greater th~n 10 microns~)) can re pond to weak g~ magnetic fie7ds ~nd magnetic field gradients; however, they tend to ~ettle rapidly, limiting their u~efulne~s for reactions requiring homogeneous condition~. Large particles also have a more limited surf~ce area per weight than smaller particles, so that less ~aterial can be c~upled to them. Examples of large particles are those of Robin~on et al. l~E~ which are 5~ 125 ~ in diameter, those of Mosb~ch and Anderson tNature~ 270:25~ 261 (1977)1 ,~ which are 6~ 140 ~ in diameter a~d th~se of Guesdon et al.
[~. Allergy Clin~ Immunol. 61~1)-2~ 27 ~1578)1 which are 5~ 160 ~ in diameter. Composite particles m~de by ~ersh and Yaverbaum lU.S. Pat. No. 3,933,997~ compri~e ferromagn~tic iron oxi~e ~Fe304) carrier particlesO
The iron oxiue carri~r particles were reported to have diameters between 1.5 ~nd 10 ~. However, based on the reported settling ra~e of 5 ~inu~es and coupling capacity of only 12 mg of protein per gram of composite particles ~L.~. ~ersh ~nd S. Yaverbaum, Clin. Chim. Acta, 63:6~ 72 (1975)], the actual size of the compositQ particles in olution is expected to be substanti~lly greater than 10 ~.
The ~ersh and Yaverbaum ferromagnetic carrier par~icles o U.S. ~. No~ 3~933,997 are ~ilanized with ~ilane~ capable Df reacting wi~h anti-digoxin ~ntibodi~s to chemically couple the antibodie~ to ~he c~rrier particles. ~ariou~ ~ilane couplin~s are di6cu~ed in U.S.
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Pat. No. 3,652,761. That the diameters of the composite particles are probably greater than 10 ~ may be explained, at least in part, by the method of silanization employed in the Hersch and Yaverbaum patent. Procedures for silanization known in the art generally differ from each other in the media chosen for the polymerization of silane and its deposition on reactive surfaces. Organic solvents such as toluene [H.W. Weetall, in: Methods is Enzymology, K. Mosbach (ed.), 44:134-148, 140 (1976~], methanol ~U.S.
pat. No. 3,933,997] and chloroform EU~S~ Pat. No. 3,652,761]
have been used. Silane depositions from aqueous alcohol and a~ueous solution with acid [H.W. Weetall, in: Methods in 15 Enzymology, su~, p. 139 (1976)], methanol ~U.S. Pat. No.

3,933,997] and chloroform [U.S. Pat. No. 3,652,761] have been used. Silane depositions from aqueous alcohol and aqueous solution with acid [H.W. Weetall, in: Methods in Enzymology, supra, p. 139 (1976)] have also been used. Each 20 of these silanization procedures employs air and/or oven drying in a dehydration step. When applied to silanization of magnetic carrier particles such dehydration methods allow the silanized surfaces of the carrier particles to contact each other, potentially resulting in interparticle bonding, 25 including, e.g., cross-linking between particles by siloxane formation, van der Waals interactions or physical adhesion between adjacent particles. This interparticle bonding yields convalently or physically bonded aggregates of silanized carrier particles of considerably larger diameter 30 than individual carrier particles. Such aggregates have low surface area per unit weight and hence, a low capacity for coupling with molecules such as antibodies, antigens or enzymes. Such aggregates also have gravitational settling times which are too short for many applications.
35 Small magnetic particles with a mean diameter in solution less than about 0.03 ~ can be kept in solution by thermal agitation and therefore do not spontaneously ~6~

~ettle. ~owev~r, the ~agnetlc field and ~agnetic field gr~dient required to r*~ove such particle~ from solution are ~o large as to reguire heavy and bulky ~agnets for their gener~tion, which are inconvenient to use ~n benc~
~ top work. ~agnets capable of geneEating ~gnetic ~ield~
in exce~s of 5000 Oer~ted~ are typi~lly required to ~eparate ~agneti~ particlel3 c3f 1~!S6 than 0. 03 1~ in diameter. An approximate quantitative relation~hip between the net force ~) acting on a particle and the magnetic field ifi given by the eguatiorl b~low (~irschbein et al-J ~
F~(Xv-X~)VH(d~/dx), where Xv and Xv are the volume susceptibilities of tbe particle and the medium, respectiYely, V is the volume of the particle, ~ i8 the applied ~agnetic ~ield and dH/ax i~ the magnetic field gradient. Th$s expression is only an approximAtion bec~use it ignores particle shape and particle interaction~. Nevertheless~ it does i~dicate that the force on a magnetic particl~ i~ directly proportional to the volume o~ the particle.
Magnetic parti~les of less th~n 0.03 ~ are used in s~ c~lled ferro~luids, which ~re described, for example, in U.S. patent No. 3,531,413. Ferr~fluids have numero~s applications, but ar~ impracti~al for appli~ations requiring ~eparation of the magnetic particles Prom ~urrounding ~edia beGau~e of the large magnetic fields and ~agneti~ ~ield gradients re~uired to effec~ the separ~tion Ferromagne~ic ~ateri~l~ in ge~eral become permanently ~agne~i2ed in repon~e to ~agnet~c field~.
Material~ termed ~cuperparamagne~ic~ experience a force in a ma~netic field gr~dient, bu~ ~o n~ b~co~e per~anently magnetiz~d. Cry tal~ o~ ~agnetic iron oxide~ ~ay be either ~errvm3gnetic or auperparamagnetic, depending on 7~

the size o~ the crystals. Superpara~gn~tic oxides s~f iron generally result when the cry~tal i~ le~ ~han about 300 A(0.03 Il) in diameter; larger cry~tal~ g~rlerally h~ve a ferromagnetic character. Following in~tia~ exposur2 to 21 magnetic field, erromagnèti~ partic:lles tend to aggr~gate be~ause of magnetic ~ttra~tion between the per~anently magnetized particl~, a~ ha,s b~en noted by Robin~on et ~1. 1.~1 and by E~er~h ~n~ Yaverb~um f~].
Di~persible magnetic irt~n oxi~e part$cle repc>Etedly having 300 A diameters and ~urface amine groups were prepared by base precipi t~tion of ;Eerrous chlc>ride and ferric chlor~de ~Fe2 /Fe3 ~1) in the presence of polyethylene iminet according to Rembaum in ~.S. Pat. No.

4,267,234. Reportedlyt these particles w~re expo ed to a magnetic field three times during preparation and were described as redispersible. The magnetiG particles were mixed with a glutaraldehyde suspension polymerization sy tem to form magnetic polyglut~raldehyde microspheres with reported diamet~r~ of 0.1 ~. Polyglutaraldehyde microspheres have coniugated aldehyde groups on the sur~ace which can form bonds to ~min~ containing molecules ~uch as protei~. Howevert in ~neral, only compounas which are capable of reacting with aldehyde groups can be directly linked to the ~urface o~ polygluthraldehyde microspheres. ~oreover, ~agne~ic polyglutarald~hyde microspheres are not ~ufficie~tly ~table for ~ertain applicati~ns.
f 2 . 2 0 SEPARATIONS IN RADIOI~MUNOASS~r S

Radioimmunoassay (PsIA) i~ a term used to describe nleth~ds for ~nalyzing . he concen'crations o~ 8ub8tances involving ~ radioacti~ely l~beled ~ubstance which binds ~o 35 an ~ntibody. The amount of radioactivity bound i altered 7~
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by the presence ~f an unl~beled test substance c~pable of binding to the ~me Dntibody. The unlDbeled ~ubstanre, i present, compete~ for binding si~es wi~h ~he l~bel~d ~ubstance ~nd thu~ decre~6e6 the amount o~ r~dioac~ivi~y bound to t~e ~ntibody. ~be decrea64 ~n bou~d radioactivity can be correlated to the ccncentrati~n of the unlabeled test substance by mean~ of a standard curve. An escenti~l ~tep of RIA i~ the ~eparat$on of bound and free label which must be accompli6hed 1n order to quantitate the bound ~r~tlon.
~ vari~ty o~ c~nventional ~eparation app~oaches have been applied to radioimmunoa~says (RIA) including coatsd tube~, p~rticulate 6y~te~6, and double ~ntibody separation methods. Coated tubes, such as described in U.S. Pat. No. 3,646~346~ allow sep~ration of bound and free label without centrifugation but suffer from two major disadvanta~es. Fir~t, the surface of the tube limits the amount of ~ntibody that can be employed in the reaction. Second the ~ntiboay i~ far remo~ed ~as much as :2 0.5 cm) from some antigen~ ~lowing the reaction betwee~
the antibody and antigen lG.~9 Pars~n~d in: Method~ in Enzymology, J. Langone (ed.) 73:225 ~1981): and P.N.
Nayak, The Ligand Quarterly 4t4):34 51981)~.
Antibodies h~ve been attached to particulate systems to facilitate ~eparation [~ee, e.g., U.S. Pats.
;~os. 3,65~,761 and 3,555,143~. Such ~ystems have large surface areas permitting nearly unlimited amounts of antibody to be used, but the particulates frequently 3D settle during th~ ass~y. The tub~ requently mu~t be agitated to w hieve even partial homogeneity ~P.~. Jacobs, The Lig2nd Quarterly, 4~4):2~ 33 tl981)J. Centri~ugation is ~till required to ef~ect complete separation o bound and ~ree label.

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,.~ --11--Antibodies may react with labeled and unlabeled molecules followed by separation using a second antibody raised to the first antibody ~Id.]. The technique, termed the double antibody method, achieves homogenPity of antibody during reaction with label but requires an incubation period for reaction of first and second antibodies followed by a centrifugation to pellet the antibodies.
Antibodies have been attached to magnetic supports in an effort to eliminate the centrifugation steps in radioimmunoassays for nortriptyline, methotrexate, digoxin, thyroxine and human placental lactogen [R.S. Kamel et al., Clin. Chem., 25(1Z):1997-2002 (1979); R.S. Kamel and J.
Gardner, Clin~ Chim. Acta, 89:363-370 (1978); U.S. Pat. No.
3,933,997; C. Dawes and J. Gardner, Clin. Chim. Acta, 86:353-356 (1978); D.S. Ithakissios _ al., Clin. Chim.
Acta, 84:69-84 (1978); D.S. Ithakissios and D.O.
Kubiatowicz, Clin. Chem. 23~11):2072-2079 (1977); and L. Nye et al., Clin. Chim Acta, 69:387-396 ~1976), respectively]. Such methods su~fer from large particle size (10-100 ~ in diameter) and require agitation to keep the antibody dispersed during the assay. Since substantial separation occurs from spontaneous settling in the absence of a magnetic field these previous methods are in fact only magnetically assisted gravimetric separations. The problem of settling was addressed by Davies and Janata whose approach in U.S. Pat. No. 4,177,253 was to employ magnetic articles comprising low density cores of 30 materials such as hollow glass or polyproplyene (4-10 ~ in diameter) with magnetic coatings (2 m~-10 ~ thick) covering a proportion of the particle surface. Anti-estradiol antibodies were coupled to such particles and their potential usefulness in estradiol RIAs -12- ~6~

W~5 demonstrated. While thi~ spproach may have overcome the problem o~ ~ettling, the particle 6ize and tbe magnetic co~ting nonethele~ present limit~tions on ~urface area and h~nce li~ata~ion~ on the availability of ~it~B for antibody couplinq.

2.3. APPLICATION O~ MAGNETIC SEPARATIONS
IN QT~ER BIOLOGICAL SYSTEMS
~ agnetic ~epar~ions hav~ been ~pplied in other biological ~yctems beside~ RIA~ SeYeral noni~otop~c im~uno~ssays, such as ~luoroi~munoassay~ (FIA) and enzym~ immunoassay~ (EIA~ have been develop~d which employ antibod~ coupled (or ~ntige~ couplffd) magnetic particles.
The principle of competitive binding i~ the ~ame in FIA
and EIA as in RIA except that fluorophores and enzymes, respectively, are ~ubstituted for radioisot~pes as l~bel.
~y way of illu~tration, ~. Pourfarzaneh et al. 2nd R.S.
Kamel et al. developed magnetiz~ble 801i~ pha~e FIAs for cortisol and phenytoin, r~pectively, utilizing f~rr~magne~ic~cellulose/iron oxid~ particles to which ~nt~bodie~ were coupled by cyanogen bromide activ~tion l~-Pourfarzaneh et al., Clin, Ch~m., 26(6):73~ 733 ~1980);
R.Sc Kamel et al., Clin. Che~., 26(9):1281-1284 (1980)~.
A no~ c~mpetiti~e ~olid pha~e ~andwich technique EIA ~or the measurement of Ig~ was d~scribed by J.-L.
Guesdon e al~ ~. Allergy Clin. Immunol,, 61(1):2~ 27 ~1978)~. By this method, anti~IgE antibodie~ coupl~d by glutaraldehyde activ~tion to magnetic polya~rylamid~
agaro~e be~ds ~re incubated with a ~e~t ~ample containing IgE to allow binding. Bound IgE i~ quantitate~ by adding a s~c~nd ~nti-~gE ~ntibody labeled with eith~r alk~line phosphatase or ~ galacto~id~e. ~he enzy~ l~beled second ~ntibody ~o~plexe~ with IgE bound to the fir~t an~ibody, forming ~he ~andwich, and the part$cle are separated magnetically. Enzyme activity associated with the particles, which is proportional to bound IgE is then measured permitting IgE quantitation.
A magnetizable solid phase non-immune radioassay for vitamin Bl2 has been reported by D~So Ithakissios and D.O. Kubiatowicz [Clin. Chem. 23(11):2072-2079 (1977)]. The principle of competitive binding in non--immune radioassays is the same as in RIA with both assays employing radioisotopic labels. However, while RIA is based on antibody-antigen binding, non-immune radioassays are based on the binding or interaction of certain biomolecules like vitamin Bl2 with specific or non-specific binding, carrier, or receptor proteins~ The magnetic particles of Ithakissios and Kubiatowicz were composed of barium ferrite particles embedded in a water-insoluble protein matxix.
In addition to their use in the solid phase biological assays just described, magnetic particles have been used for a variety of other biological purposes.
Magnetic particles have been used in cell sorting systems to 20 isolate select viruses, bacteria and other cells from mixed populations [U.S. Pats. Nos. 3,970,518; 4,230,685; and 4~267~234]o They have been used in affinity chromatography ` systems to selectively isolate and purify molecules from solution and are particularly advantageous for 25 purifications from colloidal suspensions ~K.N Mosbach and L.
Anderson, Nature 170:259-261 ~1977)]. Magnetic particles have also been used as the solid phase support in immobilized enzyme systems. Enzymes coupled to magnetic particles are contacted with substrates for a time 30 sufficient to catalyze the biochemical reaction.
Thereafter, the enzyme can be magnetically separated from products and unreac~ed substra~e and potentially can be , .. . ::

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reused. Magnetic particles have been used as supports for -chymotrypsin, R-galactosidase [U.S. Pat. No. 4,152,210], and glucose isomerase [U.S. Pat. No. 4,343,901], in immobilized enzyme systems.

3. Nomenclature The term "magnetically responsive particle" or "magnetic particle " is defined as any particle dispersible or suspendable in aqueous media without significant gravitational settling and separable from suspension by application of a magnetic field, which particle comprises a magnetic metal oxide core generally surrounded by an adsorptively or covalently bound sheath or coat bearing organic functionalities to which bioaffinity adsorbents may be covalently coupled. The term "magnetocluster" is a synonym of "magnetically responsive particle" and "magnetic particle n .
The term "metal oxide core" is defined as a crystal or group (or cluster) of crystals of a transition metal oxide having ferrospinel structure and comprising trivalent and divalent cations of the same or different transition metals~ By way of illustration, a metal oxide 25 core may be comprised of a cluster of superparamagnetic crystals of an iron oxide, or a cluster of ferromagnetic crystals of an iron oxide, or may consist of a single ferromagnetic crystal of an iron oxide.
The term "bioaffinity adsorbent" is defined 30 as any biological or other organic molecule capable of specific or nonspecific bindinq or interaction with another biological molecule, which binding or interaction may be referred to as "ligand/ligate" binding or interaction and is exemplified by, but not limited to, 7~
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antibody/antigen, antib~dy/hapten, enzyme/substrflt~, enzyme/inhibitor, enzy~e/co~actor, binding pro~ein/~ubstrate, c~ri~r protei~/ ub~tr~te9 lectin/~arbohydrate~ receptor/hor~one, receptor/effector or rep~e~sor/inducer binding~ or ~nteraction~O
The term acoupl~d ~agnetic~lly re~pon~ive particle~ or Ucoupled magnetic particle~ i8 de~ined a~ any magnetic particle to which one or more types of bioaffinity ad~orbent~ are coupled by Govaleflt bonds;
which covalent bonds ~ay be amide, e~ter~ ether ~ulfonamide, di~ulfide, ~o or other ~uitable organic linkages depending ~n the function~lities availAble for bonding on both the coat of the magnetic particle and the bio~ffinity adsorbent(~).
The term ~Eilan~ refers to any bifunctional organosilane and is de~ined ~s ~n U.S~ Pat. No. 3,652,761 ~Is an organofunction~l and ~ilico~ functional ~ilicon compound characterized in that the ilicon portion of the molecule has an ~ffinity for inorganic materials while the organic portion of the molecule i~ tailor~d to combine wi~h organicsO Silanes are ~uitable coating materi~l~ for metal oxide core~ by virtue o~ t~e~r silico~ function21ities and c~n be coupled to bioaffinity adsorbent~ through their organofunctionalities.
~he ter~ ~superparamagnetism~ is defined a~ that ~agnetic behavior exhibited by iron oxides w~th ry6tal size le~s th~n about 300 ~, which behAvior is characterized by ~e~pon~iveness to a magnetic field without resultant permanent ~agnetization.
The ~erm ~erromagnet~m~ i8 defined ~s that magnetic beha~ior exhibit~d by ir~n oxides wi~h cry~tal 8ize greater than ~bout 500 A, whi~h beha~ior i~
characteri2ed by responsiven~ to fl ~agnetic ~ield with resultant per~anent ~agnetization.

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The term ~errofluid~ is defin~d as a liquid comprising a colloid~l disper~ion o~ finely divided magnetic particles of ~ubdom~in ~ize, usually 5~ 500 ~, in a c~rrier liquid ~nd ~ ~urfactant m~teri~l, whi~h particles r~ain sub6tantially uniformly di~per~ed throughout the }iquid ca~r~er even in the Rresence of ~agnetic fi~lds of up to about 5000 Oersteds.
~ ~he term ~immunoassay~ i8 de~ined as a~y method for mea~uring the ~oncentration or a~ount of ~n analyte in 1~ a ~olution based on the immunolog$cal ~inding or interaction of a polyclonal or monoclonal ~tibody and ~n antigen, which ~ethod (a) require~ ~ ~eparation of bound from unbound analyt~; ~b) employs a radioisotopiG~
fluorometric, enzymatic, che~ilumine~cent or other label ~s the me~n~ for measuring the boun~ and/or u~bound analy~e: and ~c) may be described as ~competi~ive~ if the amount o bound ~easurable label is generally inver6ely proportional to the ~mount of analyte originally in solution or ~no~ competitive~ i~ the amount of bound 29 measurable label is generally directly proportional to the amount of analyt~ originally in solution. Tabel may be in the antigen, the anti~ody, or in d~uble ~ntibody methods, the ~econd antibody. Immunoassays ~e exemplified by~ but are not limited to, radioimmunoassays (RIA), 2 immunoradio~etric ~ssay~ ~IRMA), fluoroimmunoassays (FIA), enzyme im~unoassays ~EI~ nd ~andwich method immunoa~s~ys.
The ~erm ~bi~ding assaya or ~no~ immune assay~ i~
defined as any ~ethod f~r measuring the concentratiGn ~r amount o~ an ~nalyte i~ ~olution based on the speci~ic or nonspecific bi~ding or interaction, other than antibody/antigen biading or interaotion, ~ ~ biodf~inity ~d~orbent ~nd ~n~ther biological or organic mol~cule, which method (a) requiEes a ~eparati~n o~ bound ~rom :~z~

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unbound analyte; (b) employ6 B radioi~otopic, fluorometric~ enzymatic, chem~luminescent or other label a~ the mean~ ~or meaEuring the bound an~/or unbound ~nalyte; and (c) ~ay be de~cribed a~ acompe~ ve~ if the ~mount of bound mea~urable label i~ gen~rally ~nversely proportional to the amount of analyte origin~lly in ~olution or ~no~ competitive~ if the amount o~ bound measur~ble label i8 gener~lly directly proportional to the amount of anaIyte originally in ~olution.
The term "immobili~ed enzy~e r~action~ ifi ~efined as any enzymatically catalyzed ~iochemical conversion or ~ynthesis or degradation wherein the enzyme molecule or active ~ite thereof i8 ~ot freely 601uble but is ~dsorp~ively or covalently bound to a solid phase suppor~, which support is ~uspended in or contacted with the surr~unding medium and which may be reclaimed or ~eparated from said medium.
The term "affinity chromatography~ i5 defined as a metho~ ~or s@parating, isolating, ~ndjor puri~ying a ~elected molecule from its surrounding medium on the ba~is of it~ binding or interaction with ~ bioaffinity adsorbent adsorptively or covalently bound to ~ ~olid phase ~upport, which support i8 6uspended in or cont~cted with the surrounding medium and which may be reclaimed or ~eparated from said medium.
4. SUMMARY OF T~E INVENTION
This invention provide~ novel magnetic particles use~ul in biologic~l application~ involving the separation of molecules rom or the di~ected ~ovement of molecules in the surrounding medium. ~ethods and compo~i~ion~ ~or preparing and u~ing the magnetic particle~ ~re provided.

The magnetic particles particles comprise a magnetic metal oxide core generally surrounded by an adsorptively or covalently bound silane coat to which a wide variety of bioaffinity adsorbents c:an be covalently bonded through selected coupling chemist:ries. The magnetic metal oxide core preferably includes a group of superparamagnetic iron-oxide crystals, the coat is preferably a silane polymer and the coupling chemistries include, but are not limited to, diazotization, carbodiimide and glutaraldehyde couplings.
The magnetic particles produced by the method described herein can remain dispersed in an aqueous medium for a time sufficient to permit the particles to be used in a number of assay procedures. The particles are preferably between about 0.1 ~ and about 1.5 ~ in diameter.
Remarkably, preferred particles of the invention with mean diameters in this range can be produced with a surface ~; area as high as about 100 to 150-m2/gm, which provides a high capacity for bioaffinity adsorbent coupling.
Magnetic particles of this size range overcome the rapid settling problems of larger particles, but obviate the need for large magnets to generate the magnetic fields and magnetic field gradients re~uirèd to separate smaller particles. Magnets used to effect separations for the magnetic particles of this invention need only generate magnetic fields between about 100 and about 1000 Oersteds.
Such fields can be obtained with permanent magnets which are preferably smaller than the container which 30 holds the dispersion of magnetic particles and thus~ may be suitable for benchtop use. Although ferromagnetic particles may be useful in certain applications of the invention, particles with superparamagnetic behavior are usually preferred since superparamagnetic particles do not exhibit 35 the magnetic aggregation associated with ferromagnetic particles and permit redispersion and reuse.

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~ he method for preparing the ~agnetic particleæ
may co~prise precipit~ting met~l ~alts i~ ba~e to for~
~ine magn~tic metal oxide cry6~al~, redisp~r~ing and washing the cry~t~l~ in wat~r and in an elec~rolyte.
~agnetic ~epar~tion~ may be us~d to colle~t the crystal~
between washe~ if the cry~tal are 6uperpara~agnetic. The crystals may then be coated with a m~terial c~pable of ad orptively os covalently bonding to the metal oxide ~nd bearing orga~ic func ionalitie~ for ~oupling with ~ bioa~finity a~sorbents.
In one embodiment the coating aroun~ the metal oxide core i~ a poly~er o~ ~ilane. ~he s~lanization ~ay be performed by r~dispersing the magnetic ~etal oxide crystals in ~n acidic organic solution, ~dding an organo~ilane, dehydra~ing by heating in the presence of a wetting agent mi6cible b~th in water and the organic ~olution, and washing the resulting m~gnetic ~ilanized metal oxides. Alternatively, silanization may be performed in acidic agueous ~olution.
2~ The magnetic particles of thi~ invention can be covalently bonded by conventional coupling chemi~tries to bi~a~finity ad~orbent~ including, but not limited ~o, antibodies, antigens and 6pecif~c binding protein~, whi~h coupled magnetic particle6 an be u~ed in immunoassays or 2~ other binding asEays ~or the measurement o~ analytes in ~olution. Such a~says preerably compris~ mixing ~ s~mple conta~ning ~n unknown concentr~tion of analyte with a known a~ount of labeled analy~e in the presence of ~agnetic particle6 coupled to a bio~ffini~y ~dsorbent capable o~ binding to or interacting with both unlabeled and labeled an~lyte, Allowing the ~inding or interaction to occur, magnetically ~ep~rating the par~icles, measuring the amoun o~ label associated with the magnetic p~rticles ~nd~or the amount o~ l~bel ~ree in ~olu~ion ~d correl~ting ~he amount of label to a ~tandard curve constructed similarly to determ~ne the concentratisn of analyte in the ~ampleO
~ he ~agnetic particles o~ thiE~ invention Are ~ui~able for use in ~o~ ed ~nzyme ~y~tems, particularly wher~ enzyme recycling 1~ desir~d. ~nzymatic reactions ~re pr~fer~bly carried out by disperging Ynzym~ coupled magnetic particles in a reaction mixture containing sub~trate~), allowing the enzymatic reaction to occur, m~gnetically ~eparating the ~nzym~ coupled magn~tic particle fro~ the reaction mixture containing produc~( ) and unreacted ~ubstra~e~ ndt if de~ired, redispersing the par~icles ~n fre~h ~ubstra~e( ) thereby reusing enzyme.
Affinity chromatography ~eparations and cell ~orting car be performed using lthe magnetic particle~ of this invention, preferably by disper~ing bioaffinity ad~orbent-coupled magnetic particles in solutionæ or ~uspension~ containinq molecules or cell~ to be i~olated 2 and/or purified, ~llowing the bioaffinity ~dsoxbent ~nd the desired molecule~ or cells to interact, magnetically separ~ting the partlcle~ from the ~olutions or suspension and r~covering the isolated molecule~ or cell~ from the magnetic particles.
~t i~ furth~r c~ntempl~ted that the m~gnetic particles of this inventlon c~n be u~ed i~ ~n vivo æystems for the diagnostic ~ocaliz~tion of ~ell8 or tissu~s recognized by t~e particul~r ~ioaffinity ad~orbent coupled to the particle and ~l~o ~or magnetically directed deli~ery o~ ther~peutic age~s coupled to th~ particles to pathologi~l si~e~.
The magnetic particle~ of this invention overcome problems associated wi~h the 6ize, ~urf~ce area~
gravitation~l ~e~tling rate and magnetic character of -2.1-previously developed magnetic particles. Gravitational set~ling ti~es in exce~s of ~bout 1.5 hourB Gan be aehieved with magnetic particles of the inv~ntion, where the grav~tational ~ettling time is defin~d to be the ti~e for the t~rbidity ~ a di~per~l~n of p~lr~icle~ of the invention in be absence o~ ~ ~agnetic field to fall by ~ifty perc~nt. Magnetic ceparation times ~f le~s than ~bout ten minutes can be ~chiev~d with magnetic particies of the in~ention by contacting ~ ~es~el cont~ining disper6ion of the p~rticles with ~ pole face of ~
permanent magnet no larger in volu~e tban the volume of : the ve~sel, where the magnetic separation ti~e i~ de:Eined to b~ ~he time ~or the turbidity o~ the dispersion to fall by 95 per~ent. Furthermore, the us~ of ~ilane as the coating surrounding the metal oxide ~ore of the ~agnetic particles described herein makes po~sible the coupling of wide variety of molecules under ~n equally wide variety of coupling conditions compared to other magnetic particle coatings known in th~ art with more limited c~upling functionalitie~.
Preferred magnetically responsive particles of the invention have ~etal oxide cores comprised of clusters of superparamagnetic crystals, af~ording effic~en~
separatio~ o~ tbe particles ln ~ow ~agnetic fields (10~ 1000 Oers ed~ while maintaining ~uperpar~magnetic properties~ ~ggregati~n of particles i~ controlled during particle ~ynthesis ko produce particles which are preferab}y small enough to ~void substantial gravitational settling ov~r ti~e~ ~uf~icient to permit disper~ion~ of ~he particle~ to be used in ~n intended blological ~ssay or other ~pplication. The advantage of having superparamagnet$c cores in m~gnetically ~e~pon6i~e particles i~ that such particle can be repea~edly exposed 3 to ~agnetic fields. Because they do not be~ome ~67~9 2~

permanently ~agnetized and therefore do not ~agnetically aggreg~te, the particle~ can be redispersed ~nd reused.
Even after ~ niz~tion, preferred particle~ of the invention having cores made up of clu~ter~ of cry~tals exhibit a remarkably high 6urfæce area per uni~ weight and a generally correspondingly high ~oupli.ng capacity, which indicates that ~uch particles have an open or porou~
structure.
None of the prior art magnetic particle~ used in 1~ the biologic~l ~ystems described in Section 2 ~boYe have the same composition, ~ize, ~urfa~e are~, coupling versatility, ~ettlig propertles ~nd magne~ic behavior as the magnetic particles of the invention. The magnetic particles of this invention are suit~ble for many o~ the assays, enzyme immobili~ation, cell 60rting and a~finity chromatography procedures reported in the literature and, in fact, overcome many of the problems associated with particle ~ettling and reuse experienced in the past with - such procedures.

5~ BRIEF DESCRIPTION OF T~E FIGURES

FIG. 1 is ~ graphical representation of the change in turbidity (4 concentration) of a ~uspension of 2~ magnetic particles in the presence and ab~ence of a magnetic fiela ~5 ~ function of time.
FIG. 2 i~ a photomicrograph o~ ~uperp~ramagnetic particles ~ilanized with ~ aminopropylSrimethoxy ~ila~e.

-2~

6. DETAILED DESCRIP~ION 0~ T~E INYENTION

6.1~ M~GNETIC PARTICLE PRE~

Preferred magnetic particles of the invention may be made in two steps. ~ir~t, superpara,magnetic iron oxides are made by precipitation of divalent (Fe~ and trival2nt (Pe3~ iron ~lt~, e.g., ~eCl~ and ~eC13, in ba~e. Secondly an organosil~ne coating i8 ~pplied to _the iron oXiQ~.
The ratio o~ Fe2~ and ~e3+ can be varied without ~ubstantial changes in the ~inal product by increasing the amount of Fe2 while maintaining a ~onstant molar amount of iron. The preferred Fe2+/~e3+ ratio is 2/1 but an Fe2+/~e3+ ratio o~
4/1 al~o works ~ati~factorily in the procedure of Section

7.1 (See also Section 7~7). An ~e2~/Fe3~ ratio of 1/2 produces magnetic particles of ~lightly inferior guality to those resulting from the higher ~e2+~Fe3~ ratiOC7 This magnetic oxide tends to ~bleeda or become oluble durinq the rinsing procedure of Section 7.1 and the pazticle aize s more heterogeneous than t~e resulting from Fe2+/~e~ o~ 2/1 or 4/1. Nevertheless, it can be ~ilanized to yiel~ a usable magnetic particle as demonstrated in Section 7.7.
Aqueous ~olutions o the iron sal~s are mixed in a base such as sodium hydrsxide which results ~n the formation of a cry~alline precipitate of superparamagnetic i~n oxide. The precipit~te i8 wsshed r~pe3ted1y wi~h *a~er by magnetic211y ~eparating it ~nd redispersing it until a neu~al pH i~ re~ched. The precipitate i~ then wa~hed once in an electrolytic solution, e.q. ~ ~odium chloride ~olu~ion. ~he electrolyte wash ~tep i~ important to in~ure fineness o~

the iron oxide cry~tal~. Fin211y the precipitate i~
washed with meth~nol until a re~idue of 1.0~ (V/V3 water is left.
The repeated u~e of ~agnetic field~ to ~eparate the iron oxide from su~pen~ion dur~ng l:he ~shing ~eps is facilitated by ~uperparamagnetiæm. R~gardless of how ~any tlmes the parti~les are ~ubjected ko magnetic fields, ~hey neve~ become permanently ~agnetized ancl consequently can be redi6per~ed by mild agitat~on. Permanently magneti~ed (ferromagnetic~ ~et~l oxide& cannot be prepared by thi~
washing procedure as they tend to magnetically a~gregate after exposure to magnetic fields and cannot be homogeneous1y redi~persed.
Other divalent tran~ition metal salt~ such as magne~ium, mangane~e, cobalt, nickel, zinc and copper ~alts may be substituted for iron (II) salts in the precipitation procedure to yield magne~ic ~e~al oxides.
For example, the substitution o~ divalent cobalt chloride ~CoC12) for FeC12 in the procedure o Section 7.1 produced ferr0magnetic metal oxide particles.
Ferromagnetic ~etal oxides Luch a~ that produced with CoC12, ~ay be washed in ~he ab~ence of magnetic fields by employing conventional technigues of centrifugation or filtration between *~ hings to avoid ~agnetizing the particles. As long as the re6ulting ferromagnetic metal oxides are of su~fi~ien~ly s~all di~meter ~o remain disper~ed in aqueous ~ediat they ~ay also be il~nized and coupled ~o bio~ffinity ~dsorbents for use in fiystems reguiring a single magnetic separation, e~g. certain radioi~munoa~ay~ Ferrom3gneti6m limits particle use~ulnes~ in tho~e applic~tion~ requir$ng redisper~ion or reu~e.
Magnetic metal oxide~ produced by ba e 3 precip~ta~ion ~ay be coated by ~ny one of several suitable silanes. The silane coupling materials have two features:
They are able to adsorptively or covalently bind to the metal oxide and are able to form covalent bonds with bioaffinity adsorbents through organofunctionalities.
When silanization is used to coat the metal oxide cores of the magnetic particles of this invention, organosilanes of the general formula R~Si~OX)3 may be used wherein ~OX)3 represents a trialkoxy group, typically trimethoxy or triethoxy, and R represents any aryl or alkyl or aralkyl group terminating in aminophenyl, amino, hydroxyl, sulphydryl, aliphatic, hydrophobic or mixed function (amphipathic) or other organic group suitable for covalent coupling to a bioaffinity adsorbent. Such organosilanes include, but are not limited to, p-amino-phenyltrimethoxysilane~ 3-aminopropyltrimethoxysilane, triaminofunctional silane (H2NCH2CH2-NH-CH2CH2-NH-CH2CH2-CH2-Si-(OCH3)3, n-dodecyltriethoxysilane and n-hexyltri-methoxysilane. lFor other possible silane coupling agents see U.S. Pat. No, 3,652,761]. Generally, chlorosilanes cannot be employed unless provision is made to neutralize the hydrochloric acid evolved.
In one embodiment, the silane is deposited on the metal oxide core from acidic organic solution. The silanization reaction occurs in two steps. First, a trimethoxysilane is placed in an organic solvent, such as methanol, water and an acid, e.g., phosphorous acid o~
glacial acetic acid. It condenses to form silane polymers;

R-Si(OCH ) ~ HO ~ Si - O - Si - O -~H OH

-26- ~26~7~

Secondly, these polymers associate with the metal oxide, perhaps by forming a covalent bond with surface OH groups through dehydration:

OH OH
¦ ¦ R R
+ HO -- Si -- O ~ Si --OH OH

R R
HO ~ O -- Si -- O

Adsorption of silane polymers to the metal oxide is also 20 possible.
An important aspect of the acidic organic silanization procedure of this invention is the method of dehydration used to effect the adsorption or covalent binding of the silane polymer to the metal oxide. This 25 association is accomplished by heating the silane polymer and metal oxide in the presence of a wetting agent miscible in both the organic solvent and water. Glycerol, with a boiling poir.t of about 290C, is a suitable wetting agent. Heating to about 160-170C in ~he presence of D glycerol serves two purposes. It insures the evapora~ion of water, the or~anic solvent (which may be e.g., methanol, ethanol, dioxane, acetone or other moderately polar solvents) and any excess silane monomer. Moreover~
the presence of glycerol prevents the aggregation or 35 clumping and potential cross-linking, of particles that is an inherent problem of other silanization techniques known in the art wherein dehydration is brought about by heating to dryne 5 5 .
In another embodiment an acidic aqueous silanization procedure is used to deposit a silane polymer on the surface of the metal oxide core. Here, the metal oxide is suspended in an acidic ~pH approximately 4.5) solution of 10% silane monomer. Silanization is achieved by heating for about two hours at 90-95C. Glycerol dehydration is again used.
The presence of silane on iron oxide particles was confirmed by the following observations. First, after treatment with 6N hydrochloric acid, the iron oxide was dissolved and a white, amorphous residue was left which is not present if unsilanized iron oxide is similarly digested.
The acid insoluble residue was silane. Secondly, the diazotization method of Section 7.4 permits the attachment of antibodies to the particles. Diazotization does not promote the attachment of unsilanized particles. Finally, the attachment of antibody is extremely stable, far more stable than that resulting from the adsorption of antibodies to metal oxide~.

6.2. SILANE COUPLING CHEMISTRY
- - -An initial consideration for choosing a silane coating and the appropriate chemistry for coupling bioaffinity adsorbents to magnetic particles is the natur~
of the bioaffinity adsorbent itself, its susceptibilities to such factors as pH and temperature as well as the availability of reactive groups on the molecue for coupling. For instance, if an antibody is to be coupled to the magnetic particle, the coupling chemistry should be -28- ~2~7~

nondestructive to the immunoglobulin protein, the covalent linkage should be formed at a site on the protein molecule such that the antibody/antigen interaction will not be blocked or hindered, and the resulting linkage should be stable under the coupling conditions chosen. Similarly, if an enzyme i5 to be coupled to the magnetic particle, the coupling chemistry should not denature the enzyme protein and the covalent lin~age should be formed at a site on the molecule other than the active or catalytic site or other sites that may interfere with enzyme/substrate or enzyme/
cofactor interaction.
A variety of coupling chemistries are known in the art and have been described in U.S. Patent No. 3,652,761.
By way of illustration, diazotization can be used to couple p-aminophenyl terminated silanes to immunoglobulins. Coup-ling of immunoglobulins and other proteins to 3-aminopropyl~
terminated and N-2-aminoethyl-3-aminopropyl-terminated sil-anes has been accomplished by the use of glutaraldehyde. The procedure consists of two basic steps: l) activation of the particle by reaction with glutaraldehyde followed by removal of unreacted glutaraldehyde followed by removal and 2) reac-tion of the proteins with the activated particles followed by removal of the unreacted proteins. The procedure is widely used for the immobilization of proteins and cells lA.M.
Klibanov, Science, 219:722 (1983)]. If the magnetic particles are coated by carboxy-terminated silanes, bioaffinity adsor-bents such as proteins and immunoglobulins can be coupled to them by first treatlng the particles with 3-(3-dimethyl-aminopropyl)carbodiimlde. Generally, magnetic particles coated with silanes bearing certain organofunctionalities can be modified to -2~

substitu~e msre desirable functionali~e~ for tho e already pr~ent on the ~urface. ~o~ exampleO di~20 der~vative~ can be prepared ~ram ~ a~inopropyltriethox~
silane by reactlon wi~h p ni~r~ benzoic ~G~, reduction of the nitro group ~o ~n amine ~nd then diazot~zat$on with nitrou~ ~cid. ~he same ~ilane can be con~erted to the ~sQthiocyanoalky1silane derivative by rleaction of the ~min~ function group with thiophosgene.
To effect coupling to the ~3qnletic particle, an 0 ~queous ~olution of a bioaf~inity ~d~orbent can b~
con~acted with ~he ~ilane coa~ed par~icle at or below room temperature, Wl~en a protein (or im~u~oglobulin) is to be coupled, generaIly a r~tio of 1:10 -1:30, mg protein: mg particle i~ used. Contact periods of between about 3 to 24 hours ~re u~ually ~ufficient f~r coupling. During thi~
period, the pH is maintained at a v~lue that will not denature the bio~ffinity adsorbent and which best suits the type of linka~e being ormed, e.g. ~or azo link~ses, a p~ of ~ 9.
It has been observed that ~fter coupling of antibodies to ~ ne co~ted magnet~c particles by either the diazoti2ation, carbodii~ide, or glutaraldehyde methods de cribed in greater detail in Section 7.5, 7.8 an~ 7~10, respectiv~ly, the anti~odie~ re~ain magnetic even after 2~ the following rigorous t~eatment6s 24 hours at 50C in phosphate buffered saline ~B5)~ ~1 days at 37C in PBS~
30 minu~e~ at 23~C in IM sodium chloride, and repeated rinses in ethanol or meth~nol at r~om temperature.
~ntibodieS adsorbed to iron oxide. ~re subst~ntially 3D detached by any o~ the e treat~ent~. The~e resul~s indicate that ~he silan~ i8 ~ery tightly ~ssoci~ted with the ~etal oxide ~nd th~ ~h~ co~pllng o~ antibody to the p~rt~cle re~ults f~om an e6~entially ~rreversible covalent coupling. The tight a~sociation of ~he ~ n~ ~o the -~ 9 ~etal oxide together with the cov~lent coupling o~
bioaffinity ad~rbents (e.g~, ~ntibodie~ ~re feature~
: which impart 6tability onto coupled ~agnetic p~rticles, commercially important attribute.
s 6.3. ~SE OF MA~NE~IC PRRTICLE~
IN ~IO~OGICAL ASSAYS
, The ~agnetic particles of thi~i invention ~ay be used in immunoassays ~nd other binding a~says as defined ~n ~ection 3. The mo~t preYalent type~; ~f a~ays used for diagno~tic and research purpos~e are r~dioim~unoassay~, ~luoroimmunoassays, enzym~ immunoassay~ d no~ i~mune r3dioassays, based on the principle of ~o~pe~i~ive binding. Basically, a ligand, such a~ an antibody or specific binding protein, d~r~cted against a ligate, ~uch as an antigen, i~ ~aturated with an exce~s of labeled ligate (*ligate). lAlternatively, competi~ve a~says may be ru~ with labeled ligand and unlabeled lig~te.
No~ co~petitive a~says, s~ ~lled sandwich ~ssays, are al~o widely e~ployed.l By the method o~ this lnvention, :~ the ligand i~ coupled to a ~agnet~c particl~. ~xamples of `:~ labels are radioisotope~: tritiumr 14_c~rbon, 57-cobalt and, preferablyt 125-iodine; ~luoro~etric label~: rhodamine o~ fluoresceln isothiocyan~te; ~nd en~y~es (generally chosen fo~ the ea~e with which the enzym~tic reactlon c~n be ~ea~ured): ~lkaline phosphata~e or ~ D~galactosida ~. If nonlabel~d l~g~te i~ added to ligand along with *lig~te, le~s *ligate will be ~ound in the ligan~ ligate complex ~ the ratio of unlabeled to labeled li~At~ incr~se~ the ligan~ *ligate complex can b~ phy~ically ~ep~rated ro~ ~ligate~ the amount of unlabeled lig~te in ~ te~t ~ubstance ~n b~ de~ermined.

7~ ' .~

To ~easure unlab~led liga~e, ~ s~andard urve - must be constructed. ~hi~ i~ done by ~ixing ~ fixed amount of ligand and ~ligate ~nd ~dding ~ known ~mount of unlabeled ligate to each. When the reaction Is complete, the ligan~ ~ligate is ~epar~ted from ~l.igate. A graph i~
then made that relates the label in the ~ollected ligan~ *li~ate co~plex to the ~mount of added unlabeled l'igate, ~o determine th~ a~ount of unl~beled lig~te in an experimental ~ample, ~n 31~uot of the ~sa~ple is added to the ~ame ligan~ *ligate mixture u~ed to obtain the standard curve. The ligan~ ~ligate complex i~ collected and the label measured, ~nd the a~ount of unl~beled ligand is read from the ~tandard curve. This is possible with any ~mple, no ~atter how complex, as long as nothing interferes wi~h the ligan~ ~ligate interaction. ~y the method of this`invention, ~he ligan~ *ligate complex is separated ~agneti~ally from ree ~ligate.
This general ~ethodology can be applied in assays for the measurement of a wide variety of compounds including hor~ones, phar~acologic agents~ vitamin and cofactor~ hematological ~ub~tance~ viru~ ~ntigens, nucleic acid~, nucleotides~ glycosides and sugars. By way of illustration, th~ compounds li~ted in T~ble I ~re all measurable by magn~tic particles i~munoas~ays and binding assays lsee D. Frei~elder, Phy6ical ~ioche~istry:
~pplication~ to Biochemi~try and Mole~ul~r ~iology, p.
254, W.~. Freeman and Company~ San Prancis~o ~1976~.

6~

TABLE I

~ormone6:
Thyroid hormone~ Prolactin ~thyroxine~ tr~iod~
thyronine, thyroid Thyrocalcitonin binding globuli~, thyroi~ ~timulati~g Parathyroid hormone hormone, thyr~globulin) ~uman chorionic gonadotrophin Gastrointe~tin~l hor~nes (glucagon, g~strin, ~u~an placental lactogen enteroglucagon, ~ecretin, p~ncreoz~ Po~terior pituitary peptides min, vasoactiv~ (oxyto~in, vasopre~sin, intestinal peptide, neurophysin) gastric inhibitory pep tide, motilin, in~ulin) Bradykinin Follicl~ ~timulating hormone Cortisol Leutenizing ~ormone Corticotrophin Progesterone Human growth hormone T~stosterone E6triol Estr~diol ~ :
Digoxin Tetrahyd~ocannabinol Theophylline Barbi~ur~tes Morphine and opi~te Nicotine and met~bolic 3D alkaloid~ product~
Cardiac glycoside~ Pheno~hi~zine Prostaglandins Amphetamines Lysergic acid and deri~ativ~
-... .

-33~ 7~9 TABLE I (cont.) Vitamins and cofactors:
D, B12, folic acid, cyclic AMP

Hematolo~ical substances:
Fibrinogen, fibrin, and fibrinopeptides Prothrombin Plasminogen and plasmin Transferrin and ferritin Antihemophilic factor Erthropoietin Virus antigens: --Hepatitis antigen Polio Herpes simplex Rabies Vaccinia Q fever Several Groups A Psittacosis group arboviruses ~ ~0 Nuclei acids and nucleotides:
DN~, RNAr cytosine derivatives :
25 6.4. USE OF MAGNETIC PARTICLES IN
IMMOBILIZED ENZYME SYSTEMS
... .. _ .

Enzymes may be coupled to the magnetic particles of this invention by the methods described in Sec~ion 6.2.
They may be used in immobilized enzyme systems, particularly in batch reactors or continuous-flow stirred-tank reactors (CSTR), to facilitate separation of enzyme from product after the reaction has occurred and to permit enzyme reuse and recycle. A method for using enzyme-coupled magnetic particles in biochemical reactions was described by Dunnill and Lilly in U.S. Pat. No. 4,152,210. The magnetic particles of this invention may be advantageously ~2~
,.............................................................. .

~ubstituted or ~hose o Dunnill ~d Lilly to ~Yoid problem of ~ettling ~d to allow enzy~e recycle.
Brie~ly, sub~tr~tes are contacted with 2n2ym~ coupled ~agnetic particle~ in a reactor under condition~ of pH, temper~ture ~nd ~ub~tr~te concentration that best promote the re~ction. After completion of the reaction the ~articles are ~agnetically ~eparated from the bulk liquid (which may be a solution or ~uspen~ion) from which product can be r~trieved free of enzy~eO The enzym~ coupled 1~ ~agnetic particle~ can then ~e reused. I~mobiliz~d enzymes (coupled to no~ ~agnetic support~) have b~en used in a number o~ industri~lly important enzy~tic reac~ions, some of which ~re li~ted in Table II. ~he m~gnetic particlee of this invention can be substituted ~or the no~ magnetic ~olid phases prev~ously employed wh~ch include glass, ceramics, polyacrylamide, DEA~ cellulose, chitin, porous ~ilica, cellulose beads and alumin~ silicates.

~L~ TI
INDUSTRIALLY IMPORTANT IMMOBILIZED
EN~ ME REACTIONS
. _ _ ~, ~
Amyl~ glucosidase ~alto~e~Glucose Glucoee Oxidase Glucose/gluc~nic ~eid Glucoamylase Star~hJglucose, Dextrin/glucose ~ Amyla e St~rch/malto~e Invertase Sucrose/glucose Glucose ~o~erase Glucose/f~ucto~e ;7 Lact~se l,a~tos~e~gluco~e Trypsill Protein~ ino acid~
h~lno~cyl~e W~ acetyl- DIr ~ethionine/~ethionirle Ly60zyme Lysi~ of ~ Y ~deikticu8 I~a AFFI~IqY
~0 ., Tbe proeess of ~ff~nity ~hromatogr~phy enable~
the efficient iiol~tion o~ molecules by ~aking ul3e o~
eatures unique to ~he ~ol@cule: the ability to secognize or be recognized wi'ch ~ high degree of 6electivity by 15 bioaffini~cy ad~orbent ~uch a~ an eE~zyme or ~ntibody and the abiliky to bind or adsorb therQto. The process of af~inity chromatogr~phy s~mply involv~s placing a ~elective bloaffinity ~d~orbent or ligand in contact with a ~olution cont~ining several kinds o~ substances 20 including the desired 6pecie~, th~ ligate. ~h~ liyate is selectively ad~orbed to tbe li~and, wbic:h i~ coupled to an insoluble support or ~atrix. The nonbinding species ~re removed by wa~hing. The ligate i~ then recovered by eluting with a ~peciPie~ de~orbing ~n, e.g. ~ buf~er ~t 25 a p~ or ionic tr~ngkh ~h~t will cau~e detachment o the adsorbed ligate.
By the method o~ this invent~on, ~3gnetic par~icles may be u~ed ~18 th~ insoluble ~uppor~ to which the ligand i8 coupled. The particl~ ~ay be ~uspended in 3~ b~tch reactor~ containing the liga~e ~o be ~ol~ted. The par~icles with bound li~ate may be ~eparated magnetically froan the bulk fluid and wa~hed, ~th ~nagne~c sep~r3ti~næ
betweesl wa6hes. Iiinally, the lig~-te can be rocovered $rom the particle by de~rptlon. The magnetic p~rticle~ of this invention m~y be u~d in a variety o~ inity ~y6tem~; exempli~ied by ~ho~e listed in T~ble III.

~ 36-AFFINInr SY STEM~
S

Li~and, imlTobile enti~
Inhibitor, cofactor, prosthetic Enzymes; apoenzymes gr~up, polymeric sub~trate Enzyme Polymeric inhibito~s :: 10 Nueleic acid, ~ingle str~nd ~ucleic acid, cGmplementar~
strand ~: ~apten; antigen Antibody Antibody ~ Proteins; polysaccharides t5 ~ono~acc~laride; polysaccharide Lectins; receptor~
Lectin Gly~oproteins; receptors Small target compounds ~3inding ~rDteins Bindiny Protein Small target compounds 7. EXA~PLES
7 .1. P~aEPARATION OF METAL OX IDE

~he me'cal oxide particle~ were prepared by mixing a solution of iron~II) (F~2~) ~nd iron(III) (Fe3~J
salts wi~h base as follow~: a solution that i~ 0~ 5M
ferrous chloride (FeC12) and 0.25M gerric chloride (~eC13) (200 mls~ wa mixed with 5M ~odium hydroxide 3~ (NaO~) (200 mls) ~t 60C by pour ing both solutions into a 500 ml beaker con~aining 100 mls vf distilled water. All steps were pergor~ed ~t room ltemperA'cure unle~ o~cherwi~e indic:at@d,. The ~ixture wa~ ~tirred ~or 2 ~inutes during which time a b~ack, magne~cic precipitate i~orroedO After 7~;~

~ettling~ the volume of the 6ettled precipitate wa~
approximately 175 ml~. The concentratisn o iron oxide in he precipitate wa~ about 60 ~g/ml (ba~ed on ~ y~eld of 11.2 ~ms of iron oxid~ ~ determined in~ra)~ ~hi~ i5 in ~ontrhst to ~ommercially available ~agnlet$~ ~ron oxides, such a~ Pfizer ~228 yPe20~ (Pflzer ~ineræl~, Pigment~
and Metals Division, New York, Ne), thle standard magnetic ~xide for recording t~pe~, which ~an ~ttain concentration~
of about 700 mg/ml in aqueou~ slurry. Irhe compari~on i8 included to emphasize the f~neness o ehe parti~les ~ade by thi~ method. Very Pine particles are incapable of dense packing and ~ntrsin ~he ~o~t w~ter. L~rger and denser particlest on ~he other h~nd, pack ~e~ely, excluding the ~08t wa~er.
~he precipitate was then wash~d with water until a pH of ~ 8 was rea~Aed ~ determ~ned by p~ paperD The following washing technique was employed:

The particles were su~pended in 1.8 liters o water in a 2 liter beaker and ~ollected by magnetic extraction. The beaker was placed on top o~ a ring ~agnet, 1/2 inch hiyh ~nd 6 inche~ in di~meter, which caused the magnetic p~rticle~ to ettle. The water wa~ poured off withou~ the 1088 of particle~ by holding the ~agnet to the bottom o~
; ~ the beaker while decanting. A ~imilar w2shing technigue was employed fo~ ~11 washes through~ut, except that volumes were adju~t~d as ne~e sary. Typi~ally, three washes were ufficient to ~chieve neutral p~. The magnetic oxide w~ then wa~hed once with 1.0 li~er of 0.02M ~odium chloride ~Cl) in ~he ~me be~ker.
~ he water WDS then ~eplaced w$th methanol, lea~ing ~ trace of ~ater to cataly~e hydroly~i~ of ~he methoxy silane l~ee S~c~i~n 7.2.)~ Thi~ was acco~plished by aspirat~ng B00 ~1 of 0.2M NaCl and bringing the total ~2~ ~'69 -3~

volu~e to 1 liter with met~anol~ The ~ateri~l w~s resuspended, and ~agnetically ~xtracted; 800 ml~ of supernatant wer~ removed, and another 800 ~1~ o~ ~ethanol wer~ added. Ater three additions of ~ethanol, the oxide was ready for 6il~niz~tion in ~ ~olutilDn wb~ch w~
approximately 1% ~Y~V) water. A portion of the precipitat~ wa~ dried at 7~C for 24 hours ~nd weighed;
11.2 gram~ of magnetic iron oxide were ~ormed.
It i~ to be noted that throughout this proGedure 1~ the ~agnetic iron oxid~ particl~, because of their superparamagnetic properties, never became permanently ~agn~tized despite repeated exposure to magnetic fields.
Consequ~ntly, on~y mi}d ~gitation was reyuired to resuspend the particles during the water wafihings ~nd methanol replacement treatment.

7.2. SILANIZATION

~ he magnetic iron oxide particles (~ee S~ction 7.1.) 6u&pended in 250 mls of methanol containing approxim~tely 1% (V/Y) water were placed i~ ~ Virti~ 23 homogenizer ~Virtis Company, Inc., Gardiner, N~). Two grams of orthopho~phorous acid (~isher Scientific Co., Pittsubrgh, PA) and 10 ~1~ of ~ aminopheny1trime~hox~
~5 ~ilane (Ar7025, Petrarch Systems, Inc. t Bristol, PA) were added. In an ~lternative protocol, 5 ~1~ of glacial acetic acid hase been 6ubstituted for the 2 gms of orthophosphorGus acid. The mixture was homogenized at 23,00~ rpm f~r 10 minutes and at 9,0~ rpm for 120 ~inu~es. The contents were poured into a 500 m~ glass b~aker cont~ining 200 ml~ of gly~erol and heated on a hot pla~e un~il a ~emperature o~ 1~0-~70~C wa~ roached. ~he mixture w~s allowed to cool ~o room temperature. Both the heating and cooling ~tep~ were perfor~ed under nitrogen 1~36676~
., ~
-3~

with stirring. ~he glycerol particle slur~y (about 200 mls in volume~ wa~ poured ~nto 1.5 liter~ of water in a 2 liter beaker; the particle~ wer~ washed exhaustively tusu~lly ~our ti~es) with water ~ccording to the technique described in ~ection 7.1.
Thi~ ~ilani2ation pr~cedure ~as perormed with ~ther ~ilane6, including ~ ~minopropyltrimethox~
~ilane, ~ ~ a~inoethyl- ~ aminopropyltrimethoxy~ilane, ~ d~decyltriethoxy6ilane a~d ~ hexyltrimethoxysil~ne (~ 0800, ~ 0700, ~ 6224 and ~ 7334, respect~vely, Petrarch Sy tem~, Inc., Bri~tol, PA).
As an alternative to the ~bove ~ilanization procedure, ~ilane h~s also been ~epo~ited on ~uperparamagne~ic iron oxide (as prepared in Section 7.1) ~rom acidic aqueous solution. Superparamagnetic iron oxide with Fe2 /Fe3 ratio o$ 2 was washed w~th water as descri~ed ~n Section 7.1. The transfer to methanol was omitted. One gram o~ particles t~b~ut 20 ~ls ~f settled particle~) wa~ ~ixed with 1~0 ml~ o~ A 10~ ~olu~ion of : 20 ~ aminopropyltrimethoxy~ilane in water. The p~ was ~djusted to 4.5 with glacial acetic acid. The mixture was heated at 9~ 95C ~or 2 hours while mixing with a ~etal ~tirblade attached to an electric motor. After cooling, the par~icles were washed 3 time~ with wa~er (100 mls), 3 25 times with ~ethanol (100 ml~ ~nd 3 times with water ~100 ml~), and the pre~ence of fiilane on ~he p~rticle8 was confir~ed.

7.3. P~3YSICAL CHARACTERIST~CS OF
S I L~N I Z ED MAG NETIC PARTICLES
The mean particle diameter a~ ~easured ~y light ; sc~ttering ~nd the surf~ce area p2r gr~m as mea6ured by nitrogen gas ad~orption for E~aminophenyl ~ niz~d, 3-aminopropyl silanized, and N-2-aminoethyl-3-aminopropyl silanized particles are summarized in Table IV. The particle surface area is closely related to the capacity of the particles to bind protein; as much as 300 mg/gm of protein can be coupled to the N-2-aminoethyl-3-aminopropyl silanized particle, far higher than previously reported values of 12 mg protein/gm of particles [Hersh and Yaverbaum, Clin. Chem. Acta 63:69 (1975)]. For comparison, the surface areas per gram for two hypo-thetical spherical particles of silanized magnetite are listed in Table IV. The density of the hypothetical particles was taken to be 2.5 gm/cc, an estimate of the density of silanized magnetite particles. The diameter of each hypothetical particle was taken to be the mean diameter of the particles of the invention next to which entries for the hypothetical particle is listed. Observe that the surface area per gram of the particles of the invention as measured by nitrogen gas absorption is far gre~ter than the calculated surface area per gram for perfect spheres of silanized magnetite of the same diameter.
The greater surface area per gram of the particles of the invention indicates that the particles of the invention have a porous or otherwise open structure. Hypothetical perfect spheres of silanized magnetite having a diameter of 0.01 have calculated surface axea per gram of about 120 m2/gm.

~'~6~7~

T~L.E_ IV

CHARACTERIS~CS OF SILANI~ED ~ETIC PAR ~CLES

Mea~ured l~ypoth.
~ean Dizlm.l Surf. Ar~2 Surf. Area3 Si 1~ne ~ _ (m2/~ (m2/gm~
t~ 2 amlnoethyl- 0.561 140 4.3 minopropyl p- aminopbenyl 0.803 N~4 --3- aminopropyl 0.612 122 3.g :
-- . .

Dian,eter (in mic:rons) was measured by light ~cattering ~n a Coulter N~ 4 Particle Size Analyzer.
2 Surface area was mea~ured by 2i~2 ~as adsorption.
3 Calculated Eurf~ce area per gralD for a perf~ct phe~e with 2~ denslty at 2.5 gm/cc:.
4 Not Measured.
`- 25 ~`
- Becau~e the~ mean diam~ter~; of the ~ilar~i~ed magnetic particles produced by the proced~res of ~eotions 7.1 and 7.2 ar~ considerably ~ ller than the diameters of 30 oth~r magnetic particles de~cribed in the liter~ture, they exhibit ~slower graviJDetric 6ettling times th~n tho~e previou~ly reported. }'or instance, the settling ti~e of the particles d~scribe~ her~in i5 ~pproa~ ely 150 minu~ ir c~ntr~t to ~;ettl~ng ti~es o~O A) 5 ~inute6 for 35 the particles of ~ersh and Yaverbaum [Clin. Che~. Acta 63:
69 (1975) 1, estiJnated to b~ greate~ than 10 lJ in diameter;
and b) le58 than 1 minu~e for the particle~ of Robinson et al. [Biotech. ~io~nS~. XV:603 (1973)1 which are 50~160 )I in 6~
-4~

The ~ilanized magne~ic particl~ o~ thi~ invention ~re char~cter$~ed ~y very ~low r~ o~ grav~m@tr~c se~ling ~ a result o their ~ize and compo~ition; never-theless they re~pond promptly ~o we~k ~agnetic field~.
This ifi depict~a ln PIG. 1 where the ch~nge in turbidity over time of ~ 6u~pen ion of ~ilan~zed magnetic particles re~ulting from spontaneou particle ~ettl~ng in the absence of a magnetic field i~ comp3red So the change in the turbidity produced in ~he pre~enc~ of a ~mari~ cobalt tO ~agnet. It c~n be ~een that after 30 minute~ the turbidity of the 6uspension ha~ changed only ~lightly more than 104 in the absence of a ~agnetic field. ~o~ever, in the pr~ence o~ a we~k magnetic field, the turbidity o~ the particle su~pen ion drops by more than 95~ of it~ original value within 6 minutes. In ~nother experiment, ~ decrease in ~urbi~ity of only about 4~ in 30 minutes was observed.
A photomicrograph of ~uperp~ramagnetic particles silanized with ~ aminotrimethox~ilanes ~6IN" particles) is ~hown in F~G. 2. It can be ~en tbat the particles vary 2~ in ~hape and 8ize and that they are made up of a groups or ~lu~ters of indi~idual superparamagnetic cry~t~ls (le~s than 300 A) which appear roughly ~pherical in ~hape.

7.40 COUPLI~G OF AM~NOP~NYL M~GNETIC
PARTICLES TO A~TIBODIES TO T~ ROXINE
Fir~t~ thyroxine (~4) anti~erum w~s prepared as follow~: 5.0 ml~ of serum of ~heep immunized with T~
(obtained feom R~dioa~s~y Sy~tem~ ~aboratorie~, Inc., Carson, C~) were added ~o a 50 ml ce~trifuge ~ube. ~wo 5.0 ~1 aliquots of phosphate buffered 6~1~ne (PBS) were ~dded to the ~ube ~ollowed by 15 ~1~ of 80~ ~aturated a~monium ~ulfate, p~ 7.4, at ~C. A~t2r ~ixing, the tube was stored at 4~C for 90 mi~utes. The ~ix~ur~ was then centrifuged at 3,000 rpm for 30 minu~es a~ ~C. The ~upernatant fraction wa~ decanted and ~he pellet resu~pended and di~olv~d ~o cl~ri~y ~ 5.0 ~1~ of PBS.
The T~ ~nti~erum pr~paration (1:2 in PE~S3 wa6 di~lyzed again~t ~BS, tranæferred fro~ the dialy is tubin0 t~ a 50 ~1 ce~rifuge tube to which ~0 ml6 of ~BS were added, bringing the tot~l volume to 50 ml~. The T4 antiserum preparation ~1~10 in ~BS) wa~ refrigerated until u~ed for coupling.
To 1740 mg o~ p aminophenyl ~ilanized particles in 100 Ml o~ lN hydrochloric acid (~1), 25 ~1~ of 0.6M
3sdium nitrite (~a~021 were added. The NaN02 was ~dded ~lowl~ below the sur~ace of the particle/HCl mixture while ~aintaining th~ temperature between 0 and 5C with care taken to ~void freezing. After 10 minutes, the mixture was brought to pH 7.~ 8.5 by addition of 65 l~ls of 1.2M NaO~ and 18 mls of I~ sodium bicarbonate (Na~C03), 8till maintaining temperature a~ 0 to 5C~ . Then, 50 mls of PBS containing 100 mg of the gamma globulin fraction of 6heep ~erum containing ~ntibodie~ to thyroxine (the T~ anti~eru~ prepar~tion describ~d ~ ) were added. Th@ p~ was maintained between 7.~ 8.5 while the mixture was incubated ~or 18 hour~ at OD to 5C. The antibod~ coupled particle~ were washed exhau~tively with , O.lM ~odium phosphate bu~fer, p~ 702 (3 timefi), LM NaCl, methanol, lM NaCl and O.lM 60dium phosphate buffer again.
Wash ~teps were repeat2d twice or ~ore. All washe were . perf~rmed by di~per~ing ~he partisles and magnetical7y sep~rating them as described in sec~ion 7.1. After washing, the par~icle~ wer~ resuspended in P~S ~nd incubated overnight at 50C. The partiele~ were wa~h2d in methan~l, lM ~aCl and 0.1~ sodium pho~phate buer a~
before, ~nd ~wlce in Free T4 Tracer Buffer. The particles w~re resuspended in Free T4 Tr~cer Buf~er and ~tored at ~C u~til u~ed ~or radioimmunoass~y.

~ .~ 2~6~6~3 4~

7n50 ~AGNETIt: PAP~TICLE RADIOI3~MUNOASSA!Ir FOR THYROXINE

The quantity of antibod~ ~oup:led ~agnetic part~cle~ t~ be u~ed in the thyroxine radioimmuno~scay ~RIA) wa8 determ~ned empiric~lly using the following RIA
pracedur~:

Ten microliter~ (~lsJ of ~t~ndard were pipetted into 12x75mm polypropylene tubes followed by 500 yls of tracer and 100 ~ls of magnetic particles. After vort~xing, the ~ixture wa~ incubated ~t 37~C for 15 ~inutes ~fter which time the tubes were pl~ced on ~ magnetic ra~k for io minutes. The r~ck consisted of a te~t tube holder with a cylindrical ~button~ magnet (Incor 18, IndianR General Magnetic Product~ Corp., Valpar~iso, IN) a~ the bottom of ~ach tube. The magnetic particles with antibody and bound tracer were pulled to the ~ottom o~ ~he tubes allowing the ~nbound tracer to be removed by inverting the rack and pouring off supernat~nts. Radioactivity in th~ pellet wa~
2~ determined on a Tracor 1290 Gamma CounteE (Trazor Analytic, Inc., Elk Gro~e Vill~ge, lL).
The reagents u~ed in the as~ay were a~ follows~

Standards were p~epared by adding T4 to ~4-~ree human Qerum. T4 w~ removed fr~m the ~erum by incubation of serum wi~h activa~ed ch~rcoal followed by filtration to remove the charGo21 a~cording ~o the method of Carter ~Clin. Chem 24, 362 ~1978)~. The tracer w~s 5I-thyroxine pureha~ed from Cambridge ~edical 3D Diagnostics (~155) and was diluted into O.OLM ~ris buffer cont~ining 100 ~g/ml bovin~ ~ru~ ~lbumin, 10 ~
~alicyl~te, 50 ~g/ml ~ ~milinonaphthalen~ ~ ~u~fonic ~cid at pH 7.4. Magnetic par~icles ~t Yari~u~ conccn~rations in phosph~te buffered ~aline (PBS~ with 0.1~ bovine serum 7'~
.
~ 45-albu~nin WR e used $n the RIA to determine a suitable concentration of part~cle~ for T4 measurement~. A
quantity of ~agnetic particle6 of ~pprvxim~tely 50 ~9 per tube was c:hosen or t:he ~IA. Thi~ amount permitted good di placement oiE tracer f~om the ~ltib~dy fc~r the de~ired c:oncentration range of ~,~ (~ 32 l~g/dl) I
E~ving thu~ determined the opti~aal quantity, the P~IA procedure de~cribed ~ was performed using approximately 50 1~9 per tube of ~nagneltic particles l:o 1~ construct a radioilluDunoas&ay ~tandard curve for T4. The data obtained ~rom th RIA ~ pre~ented in Table Y.

~BLE V

RIA STANDARD CURVE FOR T
~4 T4 Concentration cpm (average o:E 2 tubes~
_.. ..
O 1~g/dl 36763 2 lJgtdl 24880 ~ 4 ~g/dl 18916

8 l~g/dl 13737 16 ~g/dl lû159 32 llg/dl 7632 ~o'cal 69219 .

7 . 6 . ~NE:TIC PARTICLE RADIOI~ aOASSI~
OR THEOPHY LLINE
Rabbit anti- theophylline antibodies we~ prepared and t:oupled to p aminophenyl silanized parti~les 3ccording to methods si~nilar to tho~e de~cribed in Sec~tion 7~ 4 . The 7~

~nti~ tlleophyllirle ai3tilbody~- coupled magnetic particles were used in ~ radioimmuno~ssay with the followirlg p~otocol: 20 ~15 of theophylline ~tzlndard ~obtained ~7y adding theophylline to theophyll1n~ free ~uman ~erura), 100 ~1~ of 125I- the~phylline tracer tobtain~d rollD C11ilical A~says, Cambrid~ nd 1 r~l of ~ntibvd~ coupled ~agnetic particles wsre vort~xed. After ~ 15 mi.nu'ce incub~tion at ~oom tempe~e~tu~e, a 10 minute magnetic separation was employed. A standard curve was c~n~tructed 3nd the data 10 obt~ined are ~hown in Table VI~

q A13LE VI

RIA STANDARD CURVE FOR T~EOP~ LLINE

The~ph,~lline Concentrzltlon ~of 2 tubes) O l~g/dl 2 ~g/dl 28217 8 l~g/al 19797 20 ~g~dl 1335Z
6û l~g/dl 814 Total 52461 7, 7. EFFECT OF VARIATION OF Fe2~/Fe3~ RATIO
OF M~AGNETIC PAR~ICLES ON T4 RADIOIMMUN0AS5P~
~
l~agnetic iron oxides were ~nade according to t!he cry6tallization procedur~ of Section 7.1 b~ maintaining c:onstant 7nolar a~ount~ o~ iron bl3t v~rying th~
Fe2+/Fe3+ ratao ~rom 4 to 9.5,. These p~rticles wese ~2~
. .
- 4~

~ilaniæ~?à, coupl~d to ~nti~ T4 antibodies and used in the T,~, RIA, a~ in ~eGtior3~ 7.2, 7.4 and 7O5~ re~pectively.
The vari~qtion of Fe2 /Fe3~ ratio did no'c 6ub~tan'ci~slly 5 affect ~he performance of the~e magn~t~c p~rticlea in the T4 RIA a~ ~hown ~t Table VI I .

T~LE VI I

T4 RIA STANDARD CURV:E:S U~SING ~NETIC
PARTICLES WIT~3 VARIED Fe ~3+ RA~IOS
T4 CDnCent:ration CDISl ~veraae of 2 tubeæ) ~e2~3~ ~,4Fe2-t~3+ ~n 5 0 ~g/dl 3563335642 1 ~Ig~dl 316Rl- 33139 2 ~g/dl 3057230195 4 ll9/dl 2470225543 ~ I~g/~l 1868019720 16 I~g/dl 128û3 11625 32 ~g/dl 10C12 B005 ~otal 77866 75636 ~ . . _ , _ 7.8. COUPLXt~G OF CARBOX~LIC ACID- TERMINATED
MA~NETlC PARTICLES 13D f31~ BINDIP~; PROTEIN
7.8.1. PREPARATIOM OF CARBO~YLIC ACID-TERMINATED M~;NETIC PARTICLES
30 - ~ - - - . . .
A ~uperparamagne~ic aron oxide was made by the procedule described ir Sec~ion 7~1 ~nd ~i.lanized ~s in Se~tion 7.2 wi~h 3- amiraopropyl~rimethoxysilang in~tead of the ~minophenyl 6il~ne. ~he alaino group of the ~ ne was then reacted with gll2taric ~nhydrid2 to ¢onvert the terminal:ion from ~n amine to c~rboxylic ~cid~, The conversion of the termination w~s accoD~pliE;hed ~
follows: five grams of amin~propyl ~ilanized particles in 5 water were washed four tlmes ~th 1.5 l~ters of O,hM
NaHC03 using ~ehe washing procedure of Sec:tion 7~1. The volume was ~djusted to 100 mls arad 2.Bri gm ~lutaric anhydride was added. ~he particles were washed two times and the reaction with glutaric ~nhydrid~ was repeated.
10 The carboxylic ~c~d- ter~inated ~agnetic particles were washed f ive ~cime-~ with water to prepare them ~or re~ction with p~te in .

7. 8. 2. C~RBODIIMID C:OUP~ OF B12 BINDI~æ PROTEIN
AND ~UMAN SE~RU~ AL13UMIN .10 CARBO~Y ~IC
ACID- ~ERMINATED M~ NE_C PART ICLES__ __ To 50 mg of carboxy- ter3r.inated magnetic particles in 1 ml of water were added 4 mq~ of 3- (3 dime~hylamin~
propyl)-carbodiimide. After mixing by shaking for 2 minutes, 0.05~mg of B12 binding ~rotein (intrinsic factor [IP~ from hog gut obtained fr~m Dr. Ro~ Allen, Denver; CO) and 0.75 mg Qf human ~eru~ albumin (~SA, obtained from Sigma Ch~mical Co., ~ 8763) were addea to ~30 ml in water. The p~ wa~ adju&ted to pH 5.6 ~nd maintained by the addi~ion of 0.1~ ~Cl or 0.1N NaO~ for three hour~. The p~rticle~ w~re then washed with 10 ml~
~f O.lM Borate with 0.5M NaCl p~ 8.3, 10 ml~ of phosphate buf~ered ~alin~ (PBS) with 0.1~ ~SA, and 10 mls of distilled water empl~ying the ~agnetic ~eparation techni~ue a in ~c~ion 7.1. P~rticles were washed three times with PBS and ~t~red in PBS unt~l u~e.

71>9~ ETIC PARl`ICLE COMPETITIVE
~1~
Usi1ng the ~P- ~nd ~ collpled ~agnetic particles made by the method of Bection 7.7, a ti'cering of the particles wa~ performed to ~cert~in the quantity of particle~ neede~ in a competitive binding ass~y for vitamin B12 (B12). The following ~s~ay prs:>tocol was u ~ed:
l00 I!ls ~f standard and 1900 IJls or' tracer buffer were added to 12x7~mm polypropyl~ne tubesc The mixture~ were -placed into a boilin~ water bath for l~ minutes to effect denatur~tion of binding proteins in hulaan ~erum samples.
15 Then l00 l l~ of v~rious concentrations of magnetic part~cles in phosphate buffer were ~dded to determine the optimal quantity of particles for assaying B12 conc~ntrations between 0 and 2000 pico~ram/ml (pg/~l).
After incubation of the mixtues for 1 bour at room temperatur~y a magnetic separation of bound and free B12 was performed acc~rding to the procedur~ of ~nd ~sing the magnetic rack described in Section 7O5~ Radioactivity in the pellet~ was then counted on a Tracor 1290 Gamma Counter ~Tracor ~nalytic, Inc. ~ Elk ~rove Village, IL).
~be reagents u~ed in the ~ssay were a~ ~oll~ws:
B12 standaras were obtained from Corning ~edical ~nd Scientific, Divi~ion o~ Corn~ng Gla~ Work~, Medfield, ~A
~474267. They ~re made with B12-free human ~erum albumin in PBS ~nd sodium azide ~dded a~ a preservative.
3D Th~ tracer was 57C~ B12 (vita~in Bl~ tagged with radioactiv~ ~obalt~ ~r~m Cornang Medic~l and ~ientific~
Division of Corning ~la~ ~ork~, ~edfield, MA, ~47~2B7.
The tracer i5 in a borate buf~er p~ 9.2, containing 0.001 potassium cyanide and ~odium ~zide. Magnetic particles &~7'~
- 5~

were diluted in PBS ~t var~vuE; concentr~tions ~ determine ~che quan~i'cy o~ par~icl~s need~d l:o mea~ure B12 concentration~ be'cween O ~nd 2000 p~/mlD
~ quantity of magnetic par~icle~ o~E approximately 50 l~g/tube wa~ selected and wa~ us~d in the Bl~
competi~ivc binding ~s~ay ~ 'co con~ltruct a ~andard c:urve; the da~a are presented in ~able VIII.

TA13LE ~JI I I
lû -B12 C~MPETITIVE BINDING RSS~__S A DARD CURVE

BConcentr~tion ~GQ9~e~es) 0 pg~ml 552 3 lûO pg/ml 5220 250 pg/ml 4169 500 pg/ml 3295 1000 pg~ml 2278 2000 pg/ml 1745 Tot~l 16515 7 .10. COUPLIPæ OF P~NETIC PARTICLES COATED W~T~
AMINOETHY Ir 3- AMINOPROE~ L SILANE TO PROTEINS
7.,10.1. CO~lPLI~æ OP' N- 2- AMINO~T~ I.- 2- AMINOPROFYL ~AGNETIC
PARTICI,ES T{~ ANTIBt)DIES ~0 T~RIIODOTHY RONINE
~0 .
Six- tenth~ of a ç~r~m of N- 2- aminoethyl- 3-aminopropyl magnetic p~rticle~ (~bbreviated "DINR
particles for ~dinitrogen", ~ignifying that th~ particles have a N/Si ra'cio of 2) prepared a~ in Section '7. 2. were ~6~7~
w51-resuspended in water. The particle~ were w~shed once in water and ~hen twice with 30 mls of O.lM pho~phate buffer, pB 7 . 4 with ~agnetic ~eparation~ between wa~hing~. After su~pending the washed p~rticle6 ~n 15 ~1~ of 0.1 phosphate, 15 ~1~ of a 5% (V/V) ~olution of glutaraldehyde, formed by dilutin~ 25~ glutar&ldehyde (G-5~82, Sigma Chemical Co., St. Loui~, ~0) with D~lM
phosphate, were added. The particles were mixed for 3 hour~ ~t room temperature by gently rotating the reaction 1~ vessel. Unreacted glutaraldehyde wa~ washed ~way with 5 additions of 30 ~1~ of O.LM pho phate bu~fer. The glutaraldehyde activated particle~ were th~n resuspended in 15 mls of 0.LM pho phate.
Trii~dothyronine ~T ) ~nti~erum ~1-6 ml8 ~5 3 obtained by immunizing rabbit~ with T3-BSA conjugate's) w~s added to ~he activated particle~ and ~tirred on a wheel mixer at room temperature for 16 to 24 hours. The ~3 antibod~ coupled particles were washed once with 3a ml~ of 0~1~ pho~phate and suspended in 15 mls of ~.2~
glycine solution in order to react any unreacted aldehyde groups. The su pension was mixed by ~hak ing ~or 25 minutesO The antibod~ coupl~d particles were wa~hed with 30 mls of 0.1 phosphate~ 30 mls of ethanol ~nd twice with 150 mls of PBS with 0.1~ bovine serum albumin (BSA~. They were resu~pended in P8S, 1% BSA and stored at ~C until used f~r RI~ for T3.

7.10.20 COUPLING OF ~ ~ AMINO~T~ ~ ~ AMINOPROPYL
MAGN~TIC PARTICLES ~0 ANTI~ODIES TO
3n T~YROID STIMULATING HORMONE
The coupling procedure of Section 6.1Q.l ~a~
followed with minor ~odificatio~sO Twenty gr~8 of DIN
particle~ were wa~hed ~hree times with 1.5 liter~ of methanol prior to glutaraldehyd~ ~tiv~tion. Glut~r-- 5~
/

aldehyde ~ctivation w~s perforll~ed ~ in ~ection 7,1U.l.
with adj~stment~ for sc2ll1e.
A goat gam~a glDbulin fra::tion c:vntalning antibodles to human thyroid stimulating hormone (TSH) wa6 5 coupled ~o the DIN particle~ r~her thlan who~e ~nti~era.
Fractionation wa~ accompli~hed by pr~c~pitatiorl of gammaglobulin$ ~ith 404 ammoni~ ulfa1ke followed by dialy~is agains~c 2BS. Approximately 4 grams of ~rotein (200 mls at 20 mg/ml) were coupled. Complete attachment 10 of protein was evident by the ab~ence o~ optical density at 280 nm in the 6upernatant after coupling. This indicated the attachment of about 20 mg of protein per gram o particl~s. Tbe particles were then washed three times with 1. 5 liter~ of lM NaCl, three times with PBS and 15 incuba'ced at 50C overnight. Par~icle~ were ~hen washed 3 more times in PBS/BSA and titered for use in the TS~ ass~y.

7011~ ~NETIC ~P~RTICLE RAD~OIMMUNOASS~
FOR TRI IODOTHY P~ONINE _ _ The quantity of particles to be used in the T3 ~IA was determined in the f~llowing a~say:

Stanaards were prep~r~d by adaing T3 to T3- free bu~nan 25 6erum a with T4 (see Section 7.5~) Tracer was 125IT3 ~rom Corning ~edical and Scientific, Divi6ion of Corning Glass Works, Medfield~ ~A (#471û6).

3~ Magnetic par~icles were diluted ~o var ious concentr~tions in P~S~ BSA to deteEmine the quantity of particles needed.

The a ~ay protocol was ~s ~ollow6: 5û ~1~ o~ ~tandard, 100 ~lls of tracer ~nd B00 ~ of D~ 7agnetic particle~
~5 were pipet ed into 12a;:75mm polypropylene tubec. After vortexing" the tubes were incuba'ced :Eor 2 hour~ at room temperature. The ~ssay was îerminat~d by IlDa~n~tic ~epar~tion. By titering the quantity of particles in the assay wlth ~ 0 ng/ml 6tand~rd/ ~ quan~i.ty of ~0 ~g/tube was deemed to be optimal or the as~ay protc>cvl~ Table ~X
shows the T3 P~IA ~tand~rd curve data obtaill~d with ~chese par t icles .

~ . IW3L~ IX

RIA STANDARD CV3~ OR T3 T3 Conc e n t r a t i on 9~e s ) 0. O ng/ml 17278 0. 25 ng/ml 1~034 0 . 50 ng/ml 13456 1. ûO ng/ml 12127 2~ 20 00 ng~ml 8758 4 . 00 ng~ml S776 8 . 00 ng/ml 3~97 ~otal 26946 ~5 . . .

7 .1~ ~ ~NETIC PARTICLE RADIOIMMUPIOASS~
FOR THY ROID ;E;TIMULATIt~ HVRMONE
3~
The ~auanti~y of particle~ to be~ usedl in the TSH
RIA was determined in the following ~ssay:

Stan~ards w~re in normal human ser~ ~Corning ~edical and 35 Scientiic, ~47186, Medfield, ~).

f~

Tracer was 125I-r~bbit ~nti-TS~ ~ntibody in PBS ~corning ~edical and Scientific, ~4741~5~ Medfield, MA).

Magnetic particle~ w~re diluted to ~eriou~ concentration~
in PB~ BSA to determine the ~uantity of particle~ need~d.

The assay protocol was as follow~: 100 yl~ of ~tandaxd and 100 ~1~ of tracer were pipetted ints 12x75 ~m polypropylene tube i vort~xed, and incubated for 3 hours 1~ ~t room tempera~ure~ ~agnetic par~icle~ ~500 ~lsl w~re added and the mixture was ~ortexe~ and inçubated for ~
hour at room temperatule~ 500 ~15 of water were adaed and the usual magnetic separation was e~ployed to ~epirate bound from unbound tracer. In the presence of TS~, a ~andwich is for~2d bet~een magnetic antibody (goat anti-TS~ antibody, ~ee Secti~n ~lO~lo) TS~ and tracer 5I-antibody (r~bbit ~nti TS~ antibody~. Thu~, increasing concentrations of ana~yte (TS~) increase the amount of bound radioactivity. Table X ~hows the T~H RIA
~tandard rurve data obtain~d by thi~ proc~dure~

TABLE X

~IA STANDARD CURVE FOR TS~

TS~ Concentration cpm O ~IU/ml* 1615 ~ U~ 309 3D 3O0 ~IU~l* 3014 6~0 ~IU/~l~ ~448 15~0 ~ 7793 30.0 ~XU/ml* 11063 60.0 ~IU/mln 15030 ~o~al 45168 ~IU=micro Int~r~ational Unit~

y` ~
^ ~

7013. CQUPLING VF MRGNETIC PARTI~LES CO~TED
WIT~ ~ ~ ~MIN~ET~Y ~ ~ AMINOPRO~ L ~ILANE
~0 EN~ MES ~ USE ~ G LUTA~ALDE~ DE
Magnetic particleæ ~1 gm3 were ~ct~v~ted with glutaraldehyde as in Sectisn 7.1001. After washing, the particles were resu~pended in 15 mls of PBS. Then 3 mls o particle~ ~2 gm) were ~ixed with 5 ~ of ~lkaline 1~ phosphat~se (Sigma Che~ical Company, ~ 9761~ or 5 ~g of galactosida~e ~Sigma Chemical Company, 5635) di~solved in 2.0 ml~ of PBS. The coupl~d particles were washed with glyci~e an~ then w~shed 5 times with PBS and re ~æpended in PBS with 0.14 ~SA.
Enzyme as~ays for magnetic alkaline phosphatase activity was perfoxmed as follows:
To a 3 ~1 cuvette 3 ~læ of 0.05~ Tri~ ~C.l were added, pH B.O, with 3mM p nitrophenyl-phosphate~ Then 100 ~ls of diluted magnetic particles with ~oupled alkaline phosphatase were added. The increase in optical density at 410 nm wa~ record~d.
Enzyme assay for magnetic ~ galactosida~e actiYity wa~ performed as follows:
To a 3ml cuvett~ 3 ml~ of O.lM phosphate were adde~, p~ 7.4, ~ith O.OlM ~erc~ptoethanol and 0.005 ~ ~itrophenyl~ ~ ~ galactopyrano~id~. ~hen 100 ~ls o~
diluted magnetic p2rticl~6 coupled to ~ galactosidase were added. The increase in optical density at 410 ~m was recorded 7 It is apparent ~ha~ many modification~ and variations of this invention a hereinabo~e ~et forth may be made without departing from the spirit ~nd 8cope thereofD ~h~ ~p~cific emb~di~ent~ de cribed are given by way nf example only and ~he invention i~ lim~ted only by the terms of th~ appended clai~s.

Claims (23)

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A magnetically-responsive particle comprising a magnetic metal oxide core generally surrounded by a silane coat to which molecules can be covalently coupled, a mass of the particles being dispersible in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field; and (b) a ninety-five-percent-turbidity-decrease sepa-ration time of less than about 10 minutes in the presence of a magnetic field;
the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

2. The magnetically-responsive particle of claim 1 wherein the metal oxide core includes a group of superpara-magnetic crystals.

3. The magnetically-responsive particle of claim 2 wherein the superparamagnetic crystals are comprised of iron oxide including divalent and trivalent iron cations.

4. The magnetically-responsive particle of claim 1 wherein the silane coat generally surrounding the core com-prises a bifunctional silane polymeric material bearing a first set of functionalities capable of adsorptively or covalently binding to the metal oxide core and a second set of functionalities capable of covalently coupling to organic molecules.

5. The magnetically-responsive particle of claim 4 wherein the silane polymeric material bear organofunctional-ities selected from the group consisting of aminophenyl, amino, carboxylic acid, hydroxyl, sulfhydryl, phenolic, ali-phatic, hydrophobic and amphipathic moieties.

6. The magnetically-responsive particle of claim 4 wherein the silane polymeric material is formed from silane monomers selected from the group consisting of p-aminophen-yltrimethoxysilane, 3-aminopropyltrimethoxysilane, N-2-amino-ethyl-3-aminopropyltrimethoxysilane, n-dodecyltrie-thoxysilane and n-hexyltrimethoxysilane.

7. The magnetically-responsive particle of claim 4 wherein the mean diameter thereof as measured by light scat-tering is between about 0.1 µ and about 1.5 µ.

8. The magnetically-responsive particle of claim 7 wherein the surface area thereof as measured by nitrogen gas adsorption is at least about 100 m2/gm.

9. A magnetically-responsive particle comprising a superparamagnetic iron oxide core generally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, the iron oxide core including a group of crystals of iron oxide, the particle having a mean diameter as measured by light scattering between about 0.1 µ and about 1.5 µ and a surface area as measured by nitrogen gas adsorption of at least about 100 m2/gm, a mass of the particles being dispers-ible in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field; and (b) a ninety-five-percent-turbidity-decrease sepa-ration time of less than about 10 minutes in the presence of a magnetic field;

the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

10. A magnetically-responsive particle comprising a ferromagnetic metal oxide core generally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, the metal oxide core including a group of crystals of metal oxide, the particle having a mean diameter as measured by light scattering between about 0.1 µ and about 1.5 µ and a surface area as measured by nitrogen gas adsorption of at least about 100 m2/gm, a mass of the particles being dispersible in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field; and (b) a ninety-five-percent-turbidity-decrease sepa-ration time of less than about 10 minutes in the presence of a magnetic field;
the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel.

11. A process for preparing magnetically-responsive particles with mean diameters between about 0.1 µ and about 1.5 µ as measured by light scattering, which process com-prises:
(a) precipitating divalent and trivalent transition metal salts in base;
(b) washing the precipitate to approximate neu-trality;

(c) washing the precipitate in an electrolyte;
(d) resuspending the precipitate in a solution of silane monomer capable of forming a polymeric coat adsorptively or covalently bound to the precipitate to which molecules can be covalently coupled; and (e) causing the silane polymeric coat to become adsorptively or covalently bound to the pre-cipitate.

12. A process for preparing superparamagnetic par-ticles with mean diameters between about 0.1 µ and about 1.5 µ as measured by light scattering, which process com-prises:
(a) precipitating divalent and trivalent iron cations of divalent and trivalent iron salts in base;
(b) washing the precipitate in water to approximate neutrality;
(c) washing the precipitate in an electrolyte; and (d) coating the washed precipitate with an adsorp-tively or covalently bound silane polymer.

13. The process of claim 12 wherein the divalent and trivalent iron salts are FeCl2 and FeCl3.

14. The process of claim 12 wherein the divalent and trivalent iron cations are used in an Fe2+/Fe3+ ratio of about 4/1 to about 1/2.

15. The process of claim 12 wherein the washings are performed by redispersing the precipitate in water and electrolyte and magnetically collecting the precipitate between washings.

16. The process of claim 12 wherein the precipitate is coated with the silane polymer by deposition of said silane polymer from acidic aqueous solution.

17. The process of claim 12 wherein the precipitate is coated with the silane polymer by deposition of said silane polymer from acidic organic solution.

18. The process of claim 17 wherein the deposition of silane polymer from acidic organic solution comprises:
(a) suspending the washed precipitate in an organic solvent containing about 1% (V/V) water;
(b) adding an acidic solution of a silane monomer;
(c) homogenizing the precipitate at high speed;
(d) mixing the precipitate with a wetting agent, which agent is miscible in the organic solvent and water;
(e) heating to a temperature sufficient to evaporate water and organic solvent; and (f) washing the wetting agent from the precipitate.

19. The process of claim 18 wherein the organic solvent is methanol and the wetting agent is glycerol.

20. The process of claim 18 wherein the solution of silane monomer is made acidic with orthophosphorous acid or glacial acetic acid.

21. The process of claim 12 wherein said silane polymer is formed from a silane monomer selected from the group consisting of p-aminophenyltrimethoxysilanel 3-amino-propyltrimethoxysilane, n-dodecyltriethoxysilane and n-hexyl-trimethoxysilane.

22. A process for preparing magnetically-responsive particles comprising superparamagnetic iron oxide cores gen-erally surrounded by a coat of polymeric silane to which molecules can be covalently coupled, which particles have mean diameters between about 0.1 µ and about 1.5 µ as measured by light scattering, a mass of the particles being dispersible in aqueous media to form an aqueous dispersion having (a) a fifty-percent-turbidity-decrease settling time of greater than about 1.5 hours in the absence of a magnetic field; and (b) a ninety-five-percent-turbidity-decrease sepa-ration time of less than about 10 minutes in the presence of a magnetic field;
the magnetic field being applied to the aqueous dispersion by bringing a vessel containing a volume of the dispersion into contact with a pole face of a permanent magnet, the permanent magnet having a volume which is less than the volume of the aqueous dispersion in the vessel, which process comprises:
(a) precipitating FeCl2 and FeCl3 in an Fe2+/Fe3+
ratio of about 2/1 with sodium hydroxide;
(b) washing the precipitate in water to approximate neutrality by redispersing and magnetically separating the precipitate;
(c) washing the precipitate in a sodium chloride solution by redispersing and magnetically separating the precipitate;
(d) suspending the washed precipitate in methanol containing about 1% (V/V) water;
(e) adding an acidic solution of a silane monomer to the suspension of precipitate;
(f) homogenizing the precipitate at high speed;
(g) mixing the precipitate with glycerol;
(h) heating the precipitate and glycerol to a temperature in the range from about 160° to about 170°C; and (i) washing glycerol from the precipitate.

23. The process of claim 22 wherein the silane monomer is selected from the group consisting of p-aminophen-yltrimethoxysilane, 3-aminopropyltrimethoxysilane, N-2-amino-ethyl.-3-aminopropyltrimethoxysilane, n-dodecyltriethoxysilane and n-hexyltrimethoxysilane.