DNA microarrays are invaluable tools for the detection and identification of nucleic acids in biosensing applications. The sensitivity and selectivity of multiplexed single-stranded DNA (ssDNA) surface bioaffinity sensing can be greatly enhanced when coupled to a surface enzymatic reaction. Herein we describe a novel method where the specific sequence-dependent adsorption of a target ssDNA template molecule onto a ssDNA-modified gold microarray is followed with the generation of multiple copies of ssRNA via in situ surface transcription by RNA polymerase. The RNA created on this “generator” element is then detected by specific adsorption onto a second adjacent “detector” element of ssDNA that is complementary to one end of the ssRNA transcript. SPR imaging is then used to detect the subsequent hybridization of complementary DNA-coated gold nanoparticles with the surface-bound RNA. This RNA transcription-based, dual element amplification method is used to detect ssDNA down to a concentration of 1 fM in a volume of 25 μL (25 zeptomoles).

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 silicacoated 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 singlestranded DNA (ssDNA) can be immobilized onto the surface of gold nanoparticles (NPs) in a single-step displacement reaction of electrostatically absorbed citrate anions. These DNAmodified 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 Murphy 3 or El Sayed 4 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 DNAfunctionalized 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: NIH Public Access Author Ma...

The effects of interparticle distance on the UV-visible absorption spectrum of gold nanocrystals aggregates in aqueous solution have been investigated. The aggregates were produced by ion-templated chelation of omega-mercaptocarboxylic acid ligands covalently attached to the nanoparticles surface. Variation of the ligand chain length provides control over the interparticle separation in the aggregates. The UV-visible spectra consist typically of a single particle band and a secondary band at higher wavelengths associated with the formation of aggregates in solution. The position of the latter depends on interparticle separation up to distances of approximately 8 nm, in accordance with existing models. Potential applications therefore include distance sensitive labels or proximity probes. Conversely, variation of the ligand length allows the preparation of nanostuctured materials with tuned optical properties.

DNA microarrays are invaluable tools for biosensing applications such as diagnostic detection of DNA and analysis of gene expression. Surface plasmon resonance imaging (SPRI) can detect unlabeled oligonucleotide targets adsorbed to the array elements. The variety of biosensing applications can be expanded by enzymatic manipulation of DNA microarray elements and the sensitivity of detection can be enhanced with the use of oligonucleotide immobilized onto a gold nanoparticle surface (detector-NP). We describe a novel method that couples a template-directed polymerase extension of a surface array element with nanoparticle-enhanced detection of the reaction product. Using this technique, it is possible to see as little as 10–100 attomoles of polymerase product representing as little as 0.25% of a monolayer. This sensitivity would allow for the detection of a specific DNA target that is present in low amounts in a sample and with partially unknown sequence. One application of this method would be to identify the presence of the aberrantly recombined DNA sequences, such as those found in the fragile sites of chromosomes.

An ex situ nanoparticle DNA detection assay utilizing DNA modified nanoparticles attached to DNA monolayer gratings on glass substrates is developed. The assay utilizes the simultaneous hybridization of a single stranded DNA (ssDNA) target molecule to both an amine-modified DNA oligonucleotide attached to an amine-reactive glass surface, and a thiol-modified DNA oligonucleotide attached to a 13 nm gold nanoparticle. Surface plasmon resonance imaging (SPRI) measurements are used to characterize the two sequential hybridization adsorption processes employed in the assay, and fluorescence microscopy is used to characterize the formation of DNA monolayer gratings via the photopatterning of the amine-reactive glass slides. First order diffraction measurements utilizing incoherent white light source and a 10 nm bandpass filter centered at 600 nm provided quantitative measurements of target ssDNA down to a concentration of 10 pM. Fourth order diffraction measurements employing a HeNe laser and avalanche photodiode were used to detect target ssDNA adsorption from 10 μL of a solution with a concentration as low as 10 fM, corresponding to 60,000 target DNA molecules. This simple yet sensitive grating-based nanoparticle DNA detection assay should be directly applicable for genetic screening, mRNA expression assays, and microRNA profiling.