Nanoparticles are nanometer-sized metallic and semiconducting particles that are the subject of extensive research in the field of nanoscale materials. Nanoparticles have potential applications in many diverse fields, including nanoscale electronic devices, multifunctional catalysts, chemical sensors, and many biological applications such as biosensors, and biological assays. Self-assembled spatial positioning of nanoparticles is a requirement for the commercial development of many of these applications.
The preparation of nanoparticles covalently bound to one or more ligands has been demonstrated by many groups with a wide variety of ligands. In order to serve as construction elements or as diagnostic devices, the precise number of ligands per nanoparticle must be known. Due to statistical nature of chemical reactions, it is very difficult to obtain particles with a defined number of ligands via direct chemical transformation. Therefore, the need exists for separation methods to isolate nanoparticles on the basis of the number of ligands affixed to the nanoparticle.
Methods of separating nanoparticle—ligand complexes are known. For example, Mirkin, et al. (Nature, Vol. 382, pg. 607, 1996) teaches the use of oligonucleotides for the synthesis of gold nanoparticles into aggregate, macroscopic clusters. Loweth, et al. (Angew. Chem., Int. Ed. Engl. 1999, Vol 38, 1808-1812) prepared dimers of phosphine stabilized gold nanoparticles using complementary strands of ssDNA bound to each particle using gel electrophoresis to isolate the dimers from the reaction mixture. Zanchet, et al. (Nano Lett. 2001, 1, 32-35) demonstrate the electrophorectic separation of gold nanoparticles on the basis of the number of bound ssDNA strands, however the authors were unable to separate particles with ligands of lengths of less than 50 bases. Niemeyer et al. (Chembiochem, 2001, 2, 260-264) prepared nanoscale networks and aggregates using biotinylated DNA and strepavidin, but did not isolate individual structures.
The above described methods for the separation and isolation of nanoparticle—ligand complexes are useful, however suffer the disadvantage of not being able to reproducibly isolate nanoparticles comprising a single species of ligand in a rapid and facile manner.
Size exclusion chromatography (SEC), also known as gel permeation chromatography, is a liquid chromatographic technique that uses a permeable support to separation analytes by size. The advantage of this separation technique is that it is rapid, easily performed, and readily lends itself to large-scale operations.
SEC has been used to characterize and separate gold nanoparticles. For example, Wei et al. (J. Chromatogr. A 836, 253-260 (1999)) described the separation of gold nanoparticles between 5 and 38 nm in size using SEC with a polymer-based column of 100 nm pore size. The shape separation of gold nanoparticles using SEC has also been described (Wei et al., Anal Chem. 71:2085-2091 (1999)).
Although the technique of SEC has a long history, to date there is no report of its use for the purpose of separating nanoparticles with a narrow size distribution having a single ligand species affixed thereto. Applicants have solved the stated problem by developing an SEC technique that permits the separation and isolation of nanopaticle-ligand complexes having a distinct ligand species.