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Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes

Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes description/claims

This application claims the benefit of priority from U.S. provisional application No. 60/393,255, filed Jul. 2, 2002, the contents of which are incorporated herein by reference in their entirety.

This invention relates to polyanionic polymer conjugates containing non-nucleotide polyanionic polymers (“polyanions”), nanoparticles bound to polyanionic polymer conjugates and methods of preparation and use thereof in detecting target analytes such as proteins or small molecules.

The binding of polyelectrolytes to gold nanoparticles for stabilization has been described (1). When mixing a polyelectrolyte solution with gold nanoparticles, the polymer composition, length, concentration, and total salt concentration all influence the coating of particles with polyelectrolytes (2). More specifically, high salt concentrations (0.05-0.1 M NaCl) have been found to cause aggregation of gold nanoparticles stabilized with polyelectrolytes (1, 2). This represents a significant limitation to coating nanoparticles with polyelectrolytes (2), and also severely limits the utility of polyanion coating technology since many applications such as biomolecule sensing require stability to electrolytes as well as temperature. For example, nucleic acid detection is typically performed in buffers that contain salt which promote nucleic acid hybridization, and biological fluids (e.g. urine) also contain elevated electrolyte concentrations which would destabilize polyelectrolyte nanoparticle complexes. Accordingly, a method for binding polyelectrolytes to nanoparticles that provides conjugates that are stable to electrolytes as well as temperature, conditions which are typically found in applications such as biomolecule sensing, would be highly desirable.

Herein we describe a method for preparing highly stable polyanion nanoparticle conjugates which utilizes a polyanion modified with a functional group to covalently attach the polyanion to the nanoparticle surface. This method creates a high density of polyanion moieties on the nanoparticle surface, providing highly stable polyanion-nanoparticle conjugates that may be used in biosensing applications.

This method differs from previous methods that utilize phosphate based nucleic acid moieties (3), which may produce unacceptable background in biosensing applications, as it is well known in the art that nucleic acids can bind to other nucleic acids, proteins, and small molecules. In the invention, the sugar and base moieties that constitute the nucleic acid have been removed, thus reducing or eliminating background in biosensing applications. This method also differs from previous strategies that utilize a monolayer of mercaptoundecanoic acid to anchor polyelectrolyte moieties through electrostatic attraction to the gold nanoparticle surface using a complicated layer-by-layer assembly technique (2). In the method described herein, the group responsible for binding to the nanoparticle surface is directly attached to the polyanion of interest for binding and the resulting conjugate may be immobilized on the particle in a single step. In addition, any ligand that may be used for biomolecule recognition may be attached to the polymer before or after nanoparticle modification.

The present invention provides polyanionic polymer conjugates containing non-nucleotide polyanions, nanoparticles bound with polyanionic polymer conjugates and methods for the synthesis of nanoparticles having polyanionic polymer conjugates attached thereto, and methods for detecting target analytes. The polyanions can be any water-soluble and water stable polymer or co-polymer with a net negative charge and at least one functional group that is able to bind to the nanoparticle surface.

More specifically, the invention provides polyanionic polymer conjugates containing non-nucleotide polyanions, nanoparticles and methods for covalent attachment of polyanionic polymers that serve either as “spacer” molecules between the surface of the nanoparticle and the recognition moiety, e.g., a recognition oligonucleotide probe, or as “filler” molecules that cover the surface of the nanoparticle between the oligonucleotide probes. These “spacer” and “filler” arrangements can allow for more efficient binding between the nanoparticle probe and biomolecule of interest. These nanoparticle probes comprising polyanionic polymers and a recogniton element are useful for biomolecule detection (e.g. nucleic acid sequence or protein), detecting protein-ligand interactions, separation of a target oligonucleotide sequence from a population of sequences, or other methods as described previously for instance in PCT/US01/10071, filed Mar. 28, 2001 and U.S. Pat. No. 6,361,944, issued Mar. 26, 2002, which are incorporated by reference in their entirety.

Incorporation of the polyanionic polymers into the nanoparticle probes increases the stability of the nanoparticles in solution, especially colloidal gold nanoparticles in high salt solutions. The use of non-nucleic acid polyanion polymers in preparing nanoparticle conjugates is advantageous for nucleic acid and protein detection because non-specific binding interference between analytes and nanoparticle conjugate probes can be reduced.

In one embodiment, the invention provides polyanionic polymer conjugates.

In another embodiment, the invention provides nanoparticles having polyanionic polymer conjugates attached thereto.

In another embodiment, the invention provides synthetic methods for the manufacture of nanoparticle probes comprising a plurality of polyanionic polymer conjugates. The polyanionic polymer conjugates may be functionalized to attach ligands or biomolecules of interest.

In another embodiment, the invention provides for methods of synthesizing nanoparticle probes having polyanionic polymers serving as spacer, or linking, molecules between the surface of the nanoparticle and oligonucleotide probe sequences.

In another embodiment, the invention provides for methods of synthesizing nanoparticle probes having polyanionic polymers serving as spacer, or linking, molecules between the surface of the nanoparticle and small molecule ligands for detecting or binding biomolecules.

In another embodiment, the invention provides for methods of synthesizing nanoparticle probes having polyanionic polymers serving as spacer, or linking, molecules between the surface of the nanoparticle and proteins for detecting or binding biomolecules.

In another embodiment, the invention provides for methods of synthesizing nanoparticle probes having polyanionic polymers serving as spacer, or linking, molecules between the surface of the nanoparticle and oligonucleotides for detecting or binding biomolecules.

In another embodiment, the invention provides for methods of synthesizing nanoparticle probes having polyanionic polymers serving as spacer, or linking, molecules between the surface of the nanoparticle and carbohydrates for detecting or binding biomolecules.

In another embodiment, the invention provides for methods of synthesizing nanoparticle probes having polyanionic polymers serving as filler molecules on the surface of the nanoparticle, between the probes (e.g. oligonucleotide, protein, etc.) that are also attached to the surface of the nanoparticle.

• Provisional Patent
• Utility Patent

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