Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chemical sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chemical composition, size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the solution an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and composition.

A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solutions, and the denaturation of these hybrid materials showed transition temperatures over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixture to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 femtomoles of an oligonucleotide.

Selective colorimetric polynucleotide detection based on Au nanoparticle probes which align in a “tail-to-tail” fashion onto a target polynucleotide is described. In this new nanoparticle-based detection system, Au particles (∼13 nm diameter), which are capped with 3‘- and 5‘-(alkanethiol)oligonucleotides, are used to complex a 24-base polynucleotide target. Hybridization of the target with the probes results in the formation of an extended polymeric Au nanoparticle/polynucleotide aggregate, which triggers a red to purple color change in solution. The color change is due to a red shift in the surface plasmon resonance of the Au nanoparticles. The aggregates exhibit characteristic, exceptionally sharp “melting transitions” (monitored at 260 or 700 nm), which allows one to distinguish target sequences that contain one base end mismatches, deletions, or an insertion from the fully complementary target. When test solutions are spotted onto a C18 reverse-phase thin-layer chromatography plate, color differentiation is enhanced and a permanent record of the test is obtained, thereby providing a better method for distinguishing the aforementioned target sequences. Significantly, one-pot colorimetric detection of the target in the presence of four strands with single base imperfections can be accomplished with this new probe system.

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.

A study aimed at understanding the factors that control the optical properties of DNA-linked gold nanoparticle aggregates containing oligonucleotide linkers of varying length (24−72 base pairs) is described. In this system, ∼15 nm diameter Au particles modified with (alkanethiol)-12 base oligomers are hybridized to a series of oligonucleotide linkers ranging from 24 to 72 base pairs (∼80−240 Å) in length. Aggregated at room temperature, the various macroscopic nanoparticle assemblies have plasmon frequency changes that are inversely dependent on the oligonucleotide linker length. Upon annealing at temperatures close to the melting temperature of the DNA, the optical properties of the DNA-linked assemblies containing the longer linkers (48 and 72 base pairs) red-shift until they are similar to the assemblies containing the shorter linkers (24 base pairs). The pre- and postannealed DNA-linked assemblies were characterized by sedimentation rate, transmission electron microscopy, dynamic light scattering, and UV−vis spectroscopy which show that the oligonucleotide linker length kinetically controls the size of the aggregates that are formed under the preannealed conditions, thereby controlling the optical properties. Through the use of small-angle X-ray scattering and electrodynamic modeling in conjunction with the techniques mentioned above, we have determined that the temperature-dependent optical changes observed upon annealing of the aggregates containing the longer oligonucleotides (48 and 72 base pairs) can be attributed to aggregate growth through an “Ostwald ripening” mechanism (where larger aggregates grow at the expense of smaller aggregates). This type of aggregate growth leads to the red-shift in plasmon frequency observed for the aggregates. Significantly, these experiments provide evidence that the optical properties of these DNA-linked nanoparticle assemblies are governed by aggregate size, regardless of oligonucleotide linker length, which has important implications for the development of colorimetric detection methods based on these nanoparticle materials.