A two-color-change, nanoparticle-based method for DNA detection
Introduction
Developing rapid DNA-detection methods is important for life science research and the clinical diagnosis of pathogenic and genetic diseases [1], [2]. Current DNA assays are dominated by techniques that rely on target hybridization using fluorescent, radioactive, or chemiluminescent molecular probes [3], [4]. Recently, gold nanoparticle probes (Au-NPs) heavily functionalized with thiolated oligonucleotides have emerged as an attractive alternative to molecular probes for nucleic acid detection [5], [6], [7]. Such probes have been used in both heterogeneous (e.g. microarrays) and homogeneous solution detection formats for the detection of short synthetic strands, [7], [8] polymerase chain-reaction products, [9] and genomic DNA targets [10], [11]. In the case of microarray-based detection, we have demonstrated the use of nanoparticle probes in scanometric, [5] SERS-based, [12], [13] and light scattering based formats [14], [15]. In many cases, these assays provide increased multiplexing capabilities, target selectivity, and sensitivity when compared to assays based upon molecular probes [12]. In the case of homogeneous formats, Au-NP probes have been used to develop simple colorimetric assays for nucleic acid targets with moderate sensitivity [7], [8], [15], [16], [17].
In a typical homogeneous assay, two sets of gold nanoparticle probes are prepared, each with a unique target-specific oligonucleotide. In the presence of complementary target strands, the Au-NP probes form target-assembled aggregates. Dissociation of these nanoparticle aggregates as a result of heating and duplex DNA melting occurs over a very narrow temperature range and is characterized by exceptionally sharp melting profiles; the full width at half-maximum (FWHM) for the first derivatives of these melting transitions can be is as low as 1 °C [8]. Sharp melting transitions allow one to differentiate perfectly complementary target strands from those with single nucleotide mismatches, [6], [7] whereas the analogous assays based upon molecular fluorophores do not offer such selectivity [7]. The formation and dissociation of nanoparticle aggregates are accompanied by color changes from red to purple and from purple to red, respectively. Such color changes can be enhanced by spotting the test solution onto a reverse-phase thin-layer chromatography (TLC) plate, known as the “Northwestern spot test”. On the plate, the color change provides a visual analysis of the state of hybridization. This spot test is moderately sensitive (100 pM), rapid, and very low cost [7], [8].
By using nanoparticles that consist of different compositions, one can monitor the hybridization state of two targets simultaneously due to the distinct optical signatures of each particle set. Specifically, Ag/Au core-shell nanoparticles can be used in an analogous spot test to provide a yellow-to-dark-brown color change in the presence of a complementary target [18]. Using pure gold and Ag/Au core-shell nanoparticle-probe systems, we have developed a new detection method for single-nucleotide-polymorphisms (SNPs). This new method relies on monitoring the two-color changes available through these two types of nanoparticle probes, leading to a dual method of identifying SNPs that is more reliable than the detection methods that are based upon a single color change. Herein, we describe the design and synthesis of Ag/Au core-shell and pure-gold-nanoparticle DNA probes with nearly identical melting profiles. We then demonstrate their use to differentiate a 30-mer target from its single-nucleotide-mutated counterpart.
Section snippets
Chemicals
AgNO3, NaBH4, HAuCl4·3H2O, and trisodium citrate were purchased from Aldrich Chemical Company. Bis(p-sulfonatophenyl)phenylphosphine (BSPP) was purchased from Strem Chemicals. Reagents required for oligonucleotide synthesis were purchased from Glen Research. TLC alumina gel RP 18 reverse-phase plates were purchased from Alltech Associates. Nanopure H2O (18.1 MΩ), purified with a Barnstead Nanopure ultrapure water system, was used for all experiments. An Eppendorf 5415C centrifuge was used for
Synthesis and stability of Ag/Au core-shell nanoparticles
Ag/Au core-shell nanoparticles were made using a two-step approach. Ag nanoparticles were sythesized in the first step, followed by shell growth in the second step. To obtain particles with ideal optical properties, in the gold-shell-growth step, one must avoid the formation of an alloy of gold and silver, the nucleation of gold clusters, and size-distribution broadening during shell growth. Gold atoms have a radius very close to that of silver atoms, and the lattice mismatch between gold and
Conclusions
This work provides a number of important observations and conclusions. First, it provides a refined method for making Ag/Au core-shell nanoparticles with the optical properties of silver but the chemical stability of gold. The conditions for gold-shell growth were optimized to minimize alloy formation, nucleation of gold particles, and the size-distribution broadening of the core-shell particles during growth. Second, Ag/Au core-shell and pure-gold nanoparticles function nearly identically as
Acknowledgments
C.A.M. acknowledges the AFOSR, DARPA, NIH, and NSF for support of this research.
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