Magneitc-nanoparticle conjugates and methods of use

Abstract

The present invention provides novel compositions of binding moiety-nanoparticle conjugates, aggregates of these conjugates, and novel methods of using these conjugates, and aggregates. The nanoparticles in these conjugates can be magnetic metal oxides, either monodisperse or polydisperse. Binding moieties can be, e.g., oligonucleotides, polypeptides, or polysaccharides. Oligonucleotide sequences are linked to either non-polymer surface functionalized metal oxides or with functionalized polymers associated with the metal oxides. The novel compositions can be used in assays for detecting target molecules, such as nucleic acids and proteins, in vitro or as magnetic resonance (MR) contrast agents to detect target molecules in living organisms.

What is claimed is:  
     
         1 . An aggregate comprising a plurality of conjugates, wherein each conjugate comprises a magnetic nanoparticle linked to a binding moiety that specifically binds to a target molecule, to another binding moiety, or to an aggregation inducing molecule, and wherein each conjugate within the aggregate is bound to at least one other conjugate in the aggregate through their respective binding moieties.  
     
     
         2 . The aggregate of  claim 1 , wherein the aggregate comprises 2 to 20 conjugates.  
     
     
         3 . The aggregate of  claim 1 , wherein the aggregate has a size of about 100 to 500 nm.  
     
     
         4 . The aggregate of  claim 1 , further comprising a target molecule to which at least two different binding moieties specifically bind.  
     
     
         5 . The aggregate of  claim 4 , wherein the target molecule is a nucleic acid, and wherein each binding moiety comprise one of two or more different oligonucleotides, wherein each oligonucleotide is complementary to a region on the target nucleic acid that is different than the regions to which the other oligonucleotides are complementary.  
     
     
         6 . The aggregate of  claim 4 , wherein the target molecule is a polypeptide, and wherein each binding moiety comprises one of two or more different antibodies, wherein each antibody binds specifically to a binding site on the polypeptide that is different than the binding sites to which the other antibodies bind.  
     
     
         7 . The aggregate of  claim 1 , further comprising an aggregation inducing molecule, and wherein the binding moieties each selectively bind to the aggregation inducing molecule.  
     
     
         8 . The aggregate of  claim 7 , wherein the aggregation inducing molecule is avidin, and the binding moieties comprise biotin.  
     
     
         9 . The aggregate of  claim 1 , wherein the binding moieties bind to each other to form the aggregate.  
     
     
         10 . The aggregate of  claim 1 , wherein each binding moiety comprises a cleavage site that is selectively cleaved by a target molecule, and wherein cleavage of the binding moiety results in separation of the conjugates and dispersal of the aggregate.  
     
     
         11 . The aggregate of  claim 10 , wherein the binding moieties are polypeptides and the target molecule is an enzyme.  
     
     
         12 . The aggregate of  claim 1 , wherein each binding moiety binds to another binding moiety to form a cleavage site that is selectively cleaved by a target molecule, and wherein cleavage of the binding moiety results in dispersal of the aggregate.  
     
     
         13 . The aggregate of  claim 12 , wherein each binding moiety comprises one of two complementary single-stranded oligonucleotides that hybridize to form a double-stranded nucleic acid comprising a cleavage site, and wherein the target molecule is an endonuclease.  
     
     
         14 . A composition comprising a mixture of at least two populations of conjugates that specifically bind to a target molecule, wherein each conjugate in the first population comprises a nanoparticle comprising a magnetic metal oxide linked to a plurality of first binding moieties that bind to a first binding site on the target molecule, and wherein each conjugate in the second population comprises a nanoparticle comprising a magnetic metal oxide linked to a plurality of second binding moieties that bind to a second binding site on the target molecule.  
     
     
         15 . The composition of  claim 14 , wherein the conjugates further comprise functional groups that link the nanoparticles to the binding moieties.  
     
     
         16 . The composition of  claim 15 , wherein the functional groups are amino, carboxy, or sulfhydryl groups.  
     
     
         17 . The composition of  claim 15 , wherein the conjugates further comprise a polymer associated with the nanoparticles, and wherein the functional groups are bound to the polymer and to the binding moieties.  
     
     
         18 . The composition of  claim 17 , wherein the polymer is hydrophilic.  
     
     
         19 . The composition of  claim 17 , wherein the polymer is a natural or synthetic polymer, or a derivative of a natural or synthetic polymer.  
     
     
         20 . The composition of  claim 17 , wherein the polymer is dextran, carboxymethyl dextran, reduced carboxymethyl dextran, crosslinked aminated dextran, or pullanan.  
     
     
         21 . The composition of  claim 17 , wherein the polymer is polyethylene glycol.  
     
     
         22 . The composition of  claim 17 , wherein the polymer is silane.  
     
     
         23 . The composition of  claim 14 , wherein the binding moieties are attached to the functional groups through disulfide bonds.  
     
     
         24 . The composition of  claim 14 , wherein the binding moieties are oligonucleotides.  
     
     
         25 . The composition of  claim 24 , wherein the oligonucleotides are attached to the nanoparticles by a single covalent bond at the 3′ or 5′ end of each oligonucleotide.  
     
     
         26 . The composition of  claim 15 , wherein the binding moieties are polypeptides.  
     
     
         27 . The composition of  claim 15 , wherein the binding moieties are antibodies.  
     
     
         28 . The composition of  claim 14 , wherein the plurality is three binding moieties per nanoparticle.  
     
     
         29 . The composition of  claim 14 , wherein the magnetic metal oxide is a superparamagnetic metal oxide.  
     
     
         30 . The composition of  claim 14 , wherein the magnetic metal oxide comprises one or more iron oxide crystals.  
     
     
         31 . The composition of  claim 14 , wherein the magnetic metal oxide has a diameter between about 1 nm and about 25 nm.  
     
     
         32 . The composition of  claim 14 , wherein the conjugate has a diameter between about 15 nm and 100 nm.  
     
     
         33 . The composition of  claim 14 , wherein the conjugate has a diameter between about 40 nm and about 60 nm.  
     
     
         34 . The composition of  claim 14 , wherein each conjugate in the composition has an R1 relaxivity between about 5 and 30 mM −1  sec −1  and an R2 relaxivity between about 15 and 100 mM −1  sec −1 .  
     
     
         35 . The composition of  claim 14 , wherein the nanoparticle is an amino-derivatized cross-linked iron oxide nanoparticle.  
     
     
         36 . A conjugate comprising a magnetic nanoparticle linked to a first binding moiety, wherein the first binding moiety comprises a cleavage site for a target molecule and specifically binds to an aggregation inducing molecule, forms a cleavage site for the target molecule when the first binding moiety binds to a second binding moiety, or specifically binds to an aggregation inducing molecule that comprises a cleavage site.  
     
     
         37 . The conjugate of  claim 36 , wherein the first binding moiety comprises a polypeptide that comprises the cleavage site, and wherein the target molecule is an enzyme.  
     
     
         38 . The conjugate of  claim 36 , wherein the first binding moiety binds to a second binding moiety to form the cleavage site that is selectively cleaved by a target molecule, and wherein the target molecule is an enzyme.  
     
     
         39 . The conjugate of  claim 38 , wherein the first and second binding moieties are complementary single-stranded oligonucleotides that hybridize to form a double-stranded nucleic acid comprising the cleavage site, and wherein the target molecule is an endonuclease.  
     
     
         40 . The conjugate of  claim 36 , wherein the first binding moiety comprises a polypeptide that contains the cleavage site and biotin, and the aggregation inducing molecule is avidin.  
     
     
         41 . The conjugate of  claim 36 , wherein the first binding moiety comprises avidin and the aggregation inducing molecule comprises biotin and the cleavage site.  
     
     
         42 . The conjugate of  claim 41 , wherein the aggregation inducing molecule comprises an oligonucleotide with a biotin molecule at each end, and the cleavage site is an internal site.  
     
     
         43 . The conjugate of  claim 41 , wherein the aggregation inducing molecule comprises a polypeptide with a biotin molecule at each end, and the cleavage site is an internal site.  
     
     
         44 . A method for determining the presence of a target molecule in a sample, the method comprising 
 obtaining a mixture of at least two populations of conjugates that specifically bind to the target molecule to form an aggregate, wherein each conjugate in the first population comprises a nanoparticle comprising a magnetic metal oxide linked to a plurality of first binding moieties that bind to a first binding site on the target molecule, and wherein each conjugate in the second population comprises a nanoparticle comprising a magnetic metal oxide linked to a plurality of second binding moieties that bind to a second binding site on the target molecule;    contacting the mixture with a fluid sample under conditions that enable the first and second binding moieties to specifically bind to any target molecules in the sample and form an aggregate of conjugates; and    determining the presence of an aggregate in the sample, wherein the presence of the aggregate indicates the presence of the target molecule.    
     
     
         45 . The method of  claim 44 , wherein the presence of an aggregate is determined by obtaining the relaxation properties of the fluid in the sample, wherein a change in the relaxation properties of the fluid indicates the presence of the target molecule.  
     
     
         46 . The method of  claim 45 , wherein a decrease in spin-spin relaxation time (T2) indicates the presence of the target molecule.  
     
     
         47 . The method of  claim 44 , wherein the target molecule is a nucleic acid, the first binding moieties are first oligonucleotides that are complementary to a first region of the target nucleic acid, and the second binding moieties are second oligonucleotides that are complementary to a second region of the target nucleic acid.  
     
     
         48 . The method of  claim 44 , wherein the target molecule is a polypeptide, the first binding moieties are first antibodies that specifically bind to a first binding site of the target polypeptide, and the second binding moieties are second antibodies that specifically bind to a second binding site of the target polypeptide.  
     
     
         49 . The method of  claim 48 , wherein the first and second antibodies are monoclonal antibodies.  
     
     
         50 . A method for determining the presence of a target molecule in a sample, the method comprising 
 obtaining one or more populations of conjugates that are capable of forming an aggregate, wherein each conjugate in a first population comprises a nanoparticle comprising a magnetic metal oxide linked to a first binding moiety, wherein the first binding moiety comprises a cleavage site for the target molecule and specifically binds to an aggregation inducing molecule, forms a cleavage site for the target molecule when the first binding moiety binds to a second binding moiety in a second population of conjugates, or specifically binds to an aggregation inducing molecule that comprises a cleavage site;    mixing the conjugates of the one or more populations in a fluid under conditions that enable the binding moieties to specifically bind to each other or to an aggregation inducing molecule to form aggregates in the fluid;    mixing the fluid containing the aggregates with a fluid sample under conditions that enable any target molecules in the sample to cleave the cleavage sites in the aggregates; and    determining the presence of aggregates in the sample, wherein the absence of aggregates indicates the presence of the target molecule.    
     
     
         51 . The method of  claim 50 , wherein the absence of aggregates is determined by obtaining the relaxation properties of the fluid in the sample, wherein a change in the relaxation properties of the fluid indicates the presence of the target molecule.  
     
     
         52 . The method of  claim 51 , wherein an increase in spin-spin relaxation time (T2) indicates the presence of the target molecule.  
     
     
         53 . The method of  claim 50 , wherein the target molecule is an enzyme, and the first binding moiety comprises a polypeptide that contains the cleavage site.  
     
     
         54 . The method of  claim 53 , wherein the binding moieties comprise a polypeptide that contains the cleavage site and biotin, and the aggregation inducing molecule comprises avidin.  
     
     
         55 . The method of  claim 50 , wherein the target molecule is an endonuclease, and the first and second binding moieties are complementary single-stranded oligonucleotides that hybridize to form a double-stranded nucleic acid comprising the cleavage site selectively cleaved by the endonuclease.  
     
     
         56 . The method of  claim 50 , wherein the first binding moiety comprises avidin and the aggregation inducing molecule comprises biotin and the cleavage site.  
     
     
         57 . The method of  claim 56 , wherein the aggregation inducing molecule comprises an oligonucleotide with a biotin molecule at each end, and the cleavage site is an internal site.  
     
     
         58 . The method of  claim 56 , wherein the aggregation inducing molecule comprises a polypeptide with a biotin molecule at each end, and the cleavage site is an internal site.  
     
     
         59 . The method of  claim 50 , wherein the aggregation inducing molecule is an antibody.  
     
     
         60 . A method for determining the presence of a target molecule in a sample, the method comprising 
 obtaining first and second populations of oligonucleotide-nanoparticle conjugates, wherein each conjugate in the first population comprises a nanoparticle having a magnetic metal oxide associated with a polymer having functional groups; and a plurality of first oligonucleotides attached to the functional groups on the nanoparticle; and wherein each conjugate in the second population comprises a nanoparticle having a metal oxide associated with a polymer having functional groups; and a plurality of second oligonucleotides attached to the functional groups on the nanoparticle; wherein the first and second oligonucleotides are each complementary to first and second portions of the target nucleic acid, and wherein the oligonucleotides in each population are the same on each conjugate in the population and different than the oligonucleotides on the conjugates in the other populations;    preparing a mixture of the first and second populations of oligonucleotide-nanoparticle conjugates;    obtaining a fluid sample;    contacting the mixture with the sample under conditions that enable any target nucleic acid in the sample to hybridize to the first and second oligonucleotides of both populations of conjugates; and    obtaining the relaxation properties of the fluid in the sample, wherein a change in the relaxation properties of the fluid indicates the presence of the target nucleic acid.    
     
     
         61 . An assay for determining aggregate formation in a fluid sample, the method comprising 
 adding the composition of  claim 14  to the fluid sample under conditions that enable aggregate formation;    measuring the relaxation properties of the sample over time after addition of the composition, wherein a decrease in spin-spin relaxation (T2) indicates aggregate formation.    
     
     
         62 . An assay for determining aggregate dispersal in a fluid sample, the method comprising 
 adding an aggregate formed from the conjugate of  claim 36  to the fluid sample under conditions that enable cleavage of the aggregate;    measuring the relaxation properties of the sample over time after addition of the conjugate, wherein an increase in spin-spin relaxation (T2) indicates aggregate dispersal.    
     
     
         63 . The method of  claim 44 , wherein the fluid is optically transparent.  
     
     
         64 . The method of  claim 44 , wherein the fluid is optically translucent.  
     
     
         65 . The method of  claim 44 , wherein the fluid is optically turbid.  
     
     
         66 . The method of  claim 44 , wherein the fluid is optically opaque.  
     
     
         67 . The method of  claim 44 , wherein the fluid is water, saline, buffered saline, or a biological fluid.  
     
     
         68 . The method of  claim 67 , wherein the biological fluid is blood, a cell homogenate, a tissue homogenate, a cell extract, a tissue extract, a cell suspension, a tissue suspension, milk, urine, saliva, semen, or spinal fluid.  
     
     
         69 . The method of  claim 44 , wherein the amount of change in the relaxation properties indicates a concentration of target molecules in the sample.  
     
     
         70 . The method of  claim 44 , wherein the magnetic metal oxide is a superparamagnetic metal oxide.  
     
     
         71 . The method of  claim 70 , wherein the superparamagnetic metal oxide comprises iron oxide.  
     
     
         72 . The method of  claim 44 , wherein the metal oxides are monodisperse.  
     
     
         73 . The method of  claim 44 , wherein the metal oxides are polydisperse.  
     
     
         74 . A method for purifying a target molecule from a sample, the method comprising 
 obtaining a conjugate comprising a nanoparticle comprising a magnetic metal oxide linked by a cleavable bond to a binding moiety that specifically binds to a binding site on the target molecule;    obtaining a sample containing the target molecule in a fluid;    mixing the conjugates with the sample under conditions sufficient to enable target molecules in the sample to bind to the binding moiety on the conjugate to form target molecule-binding moiety complexes;    separating the conjugates from the sample; and    cleaving the cleavable bond to separate the target molecule-binding moiety complexes from the conjugates, thereby purifying the target molecules.    
     
     
         75 . The method of  claim 74 , wherein the target molecule is a nucleic acid, and the binding moiety is an oligonucleotide that is complementary to a portion of the target nucleic acid.  
     
     
         76 . The method of  claim 74 , wherein the target molecule is a polypeptide, and the binding moiety is an antibody that specifically binds to a portion of the target polypeptide.  
     
     
         77 . An assay for simultaneously determining the presence of a target nucleic acid in a plurality of samples, the method comprising 
 obtaining first and second populations of oligonucleotide-nanoparticle conjugates, wherein each conjugate in the first population comprises a nanoparticle having a magnetic metal oxide associated with a polymer having functional groups; and a plurality of first oligonucleotides attached to the functional groups on the nanoparticle, and wherein each conjugate in the second population comprises a nanoparticle having a metal oxide associated with a polymer having functional groups; and a plurality of second oligonucleotides attached to the functional groups on the nanoparticle; wherein the first and second oligonucleotides are each complementary to first and second portions of the target nucleic acid, and wherein the oligonucleotides in each population are the same on each conjugate in the population and different than the oligonucleotides on the conjugates in the other populations;    preparing a mixture of the first and second populations of oligonucleotide-nanoparticle conjugates;    obtaining a plurality of fluid samples;    contacting a portion of the mixture with each of the plurality of samples under conditions that enable any target nucleic acid in the samples to hybridize to the first and second oligonucleotides of both populations of oligonucleotide-nanoparticle conjugates; and    simultaneously obtaining the relaxation properties of the fluid in each of the plurality of samples, wherein a change in the relaxation properties of a sample indicates the presence of the target nucleic acid in that sample.    
     
     
         78 . The method of  claim 77 , wherein the amount of change in the relaxation properties of a sample indicates a concentration of target nucleic acid in that sample.  
     
     
         79 . A method for determining the presence of a target molecule in a subject, the method comprising: 
 administering to the subject at least one population of conjugates, wherein each conjugate comprises a nanoparticle having a magnetic metal oxide linked to a binding moiety that specifically binds to the target molecule;    providing sufficient time for the binding moieties to bind to target molecules in the subject; and    generating a magnetic resonance (MR) image of the subject, wherein a signal in the image indicates the presence of a target molecule.    
     
     
         80 . The method of  claim 79 , wherein the target molecule is a nucleic acid, and the binding moiety is an oligonucleotide that is complementary to a portion of the target nucleic acid.  
     
     
         81 . The method of  claim 79 , wherein the target molecule is a protein, and the binding moiety is an antibody that specifically binds to the target molecule.  
     
     
         82 . The method of  claim 80 , wherein at least two populations of conjugates are administered to the subject, wherein the binding moieties on the conjugates in each population are identical within the population and different from the binding moieties on the conjugates in other populations; and wherein the binding moieties in different populations of conjugates specifically bind to different portions of the target molecule.