Magnetic-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.

1 .- 10 . (canceled) 
     
     
         11 . A method for determining the presence of a target molecule in a sample, the method comprising:
 (i) providing one or more aggregates in a fluid, wherein the aggregates comprise (a) nanoparticles comprising a magnetic metal oxide linked to a binding moiety, and (b) an aggregation inducing molecule,   wherein the binding moiety comprises a cleavage site for the target molecule and specifically binds to the aggregation inducing molecule, or wherein the aggregation inducing molecule comprises a cleavage site for the target molecule and specifically binds to the binding moiety;   (ii) following step (i), combining the fluid and the sample under conditions that enable a target molecule in the sample to cleave the cleavage site; and   (iii) following step (ii), determining whether a target molecule in the sample cleaved the cleavage site by measuring the spin-spin (T2) relaxation time of the fluid, wherein an increase in T2 relaxation times indicates the presence of the target molecule in the sample.   
     
     
         12 . The method of  claim 11 , wherein the target molecule is an enzyme, and the binding moiety comprises a polypeptide that contains the cleavage site. 
     
     
         13 . The method of  claim 12 , wherein the binding moiety comprises a polypeptide that contains the cleavage site and biotin, and the aggregation inducing molecule comprises avidin. 
     
     
         14 . The method of  claim 11 , wherein the target molecule is an endonuclease, and the binding moiety and aggregation inducing molecule are complementary single-stranded oligonucleotides that hybridize to form a double-stranded nucleic acid comprising the cleavage site selectively cleaved by the endonuclease. 
     
     
         15 . The method of  claim 11 , wherein the binding moiety comprises avidin and the aggregation inducing molecule comprises biotin and the cleavage site. 
     
     
         16 . The method of  claim 15 , wherein the aggregation inducing molecule comprises an oligonucleotide with a biotin molecule at each end, and the cleavage site is an internal site. 
     
     
         17 . The method of  claim 15 , wherein the aggregation inducing molecule comprises a polypeptide with a biotin molecule at each end, and the cleavage site is an internal site.