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A novel method for preparing gold nanorods that are first coated with a thin silica film and then functionalized with single stranded DNA (ssDNA) is presented. Coating the nanorods with 3-5 nm of silica improves their solubility and stability. Amine-modified ssDNA is attached to the silica-coated gold nanorods via a reductive amination reaction with an aldehyde trimethoxysilane monolayer. The nanorods exhibit an intense absorption band at 780 nm, and are used to enhance the sensitivity of surface plasmon resonance imaging (SPRI) measurements on DNA microarrays.
Metallic nanorods are nanoscale materials that possess unique optical and electronic properties which make them extremely useful when incorporated into schemes for the detection of biomolecules such as DNA, RNA and proteins. To successfully integrate these materials into bioaffinity detection assays, the nanoscale surfaces must first be functionalized with biomolecules without altering their stability in solution. For example, thiol-modified single-stranded DNA (ssDNA) can be immobilized onto the surface of gold nanoparticles (NPs) in a single-step displacement reaction of electrostatically absorbed citrate anions. These DNA-modified NPs, first reported by Mirkin et al.1 and Alivisatos et al.2 have been used extensively for the detection and identification of oligonucleotides. The straightforward thiol attachment chemistry is made possible by the anionic character of the nanoparticle surface due to the presence of the citrate. In contrast, gold nanorods produced by the methods developed by either Murphy3 or El Sayed4 have a net positive surface charge due to the presence of an adsorbed monolayer of the surfactant, hexadecyltrimethyl-ammonium bromide (CTAB), on the nanorod surface. Thus, the thiol chemistry used to modify gold NPs is very difficult when employed for the attachment of ssDNA to surfactant-coated gold nanorods. The reasons for this are that the high density of the surfactant monolayer decreases the access of the thiol-modified ssDNA to the nanorod surface and the negatively-charged phosphate backbone of the ssDNA interacts with the positively charged CTAB molecules; the net result is typically a rapid aggregation and precipitation of the gold nanorods from solution. This letter describes an alternative strategy for preparing ssDNA-functionalized gold nanorods based on a multi-step process in which the gold nanorods are first modified with a thin silica film and then the ssDNA is attached to the silica shell via an aldehyde coupling reaction. We further demonstrate that these DNA-functionalized silica-coated gold nanorods can be used to greatly enhance the sensitivity of surface plasmon resonance imaging (SPRI) measurements of DNA hybridization adsorption onto DNA microarrays.
The preparation of DNA-functionalized silica-coated gold nanorods requires a sequential surface modification process that is shown schematically in Figure 1. The functionalized gold nanorod synthesis can be divided into three main steps:
When an equimolar mixture of A25 and T25 DNA-functionalized silica-coated gold nanorods were allowed to react at room temperature, these aggregated within an hour. Both, transversal and longitudinal surface plasmon absorption bands at 517 and 780 nm in the UV-visible absorption spectra (Supporting Information, Figure S2) decreased in intensity due to hybridization of the complementary nanorods resulting in aggregation and eventual loss of the nanorods from solution by precipitation. The nanorods aggregation was also indicated by TEM imaging (Supporting Information, inset in Figure S2).
The application of DNA-functionalized silica-coated gold nanorods to enhance SPRI measurements was demonstrated by the sequence specific adsorption of gold nanorods onto a DNA microarray. Briefly, a two component ssDNA microarray was created on a set of 16 gold thin film spots (1 mm diameter, 45 nm thickness) on an SF10 glass slide. The details of the DNA microarray fabrication process and surface attachment chemistry have been published previously elsewhere.14-15 Two sequences were used in the DNA microarray: T25 and A25. Exposure of the microarray to a solution of T25 DNA-modified silica-coated gold nanorods in an SPRimager instrument and flow cell (GWC Technologies) for approximately 10 min. led to the SPRI differential reflectivity image and line profile shown in Figure 3. A very large differential reflectivity change (Δ%R = 28 ± 2.6%) was observed due to the hybridization adsorption of the T25 gold nanorods onto the A25 DNA microarray elements. This large increase in the Δ%R is due to the strong optical properties of the nanorods, which are governed by the wavelength dependent complex refractive index of the metal. The adsorption of the gold nanorods produces large changes in the local electromagnetic fields in the vicinity of the interface. The reflectivity change observed for the adsorption of a full monolayer of gold nanorods was comparable to the value previously reported for the adsorption of a full monolayer of gold nanoparticles16 and was approximately 15 times larger than the response observed for the hybridization adsorption of a full monolayer of DNA. The optical response from a dilute monolayer of gold nanorods is expected to differ from the optical response of a dilute monolayer of gold nanoparticles due to the differences in their optical properties; a full study of the wavelength and surface coverage dependence of these optical properties will be detailed in a later paper. Non-specific adsorption of the nanorods onto the remaining T25 elements was minimum.
In summary, the experiments reported here show that silica-coated gold nanorods modified with an aldehyde silane monolayer can be successfully reacted with amine-terminated ssDNA via a reductive amination reaction to create stable solutions of DNA-functionalized silica-coated gold nanorods. Additionally, the DNA-functionalized gold nanorods are capable of hybridization with the complementary DNA either immobilized onto a planar gold surface or attached to another nanorod. Future experiments will employ DNA-functionalized gold nanorods in conjunction with enzymatic amplification methods for the ultrasensitive detection of DNA and RNA with nanorod-enhanced SPRI.
This work was supported by grants from the National Institute of Health (GM-059622) and the National Science Foundation (CHE-0551935). The authors are grateful to the California Institute for Telecommunications and Information Technology at UCI for help in the use of TEM instrumentation, and to Dr. Lida K. Gifford for useful discussions. RMC has a financial interest in GWC Technologies.