Gold nanoparticle probe-based gene expression analysis with unamplified total human RNA

Abstract

Microarray-based gene expression analysis plays a pivotal role in modern biology and is poised to enter the field of molecular diagnostics. Current microarray-based gene expression systems typically require enzymatic conversion of mRNA into labeled cDNA or cRNA. Conversion to cRNA involves a target amplification step that overcomes the low sensitivity associated with commonly used fluorescent detection methods. Herein, we present a novel enzyme-free, microarray-based gene expression system that uses unamplified total human RNA sample as the target nucleic acid. The detection of microarray-bound RNA molecules is accomplished by targeting the poly-A tail with an oligo-dT20 modified gold nanoparticle probe, signal amplification by autometallography, and subsequent measurement of nanoparticle-mediated light scattering. The high sensitivity afforded by the nanoparticle probes allows differential gene expression from as little as 0.5 microg unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps.

Optical microarray biosensing techniques

“…An evanescent field generated at the surface of a bulk waveguide by multiple TIRF has also been used to induce light scattering. This can be accomplished using a simple setup such as the one proposed by Nanosphere (Northbrook, IL), 103 wherein side illumination by either red light-emitting diodes 60 or white light 65 is used to induce the scattering of silveramplified gold nanoparticles. The scattered light is then focused and detected on a photosensor.…”

“…This technology has also been combined with the sandwich-assay approach illustrated in Fig. 5 to analyze expression levels in total human RNA 60 and to detect SNPs. 65 The detection involved a secondary gold colloid tagged oligonucleotide added after target hybridization, and signal amplification was then achieved by silver deposition on the nanoparticles.…”

Electrochemical genosensors for biomedical applications based on gold nanoparticles

“…Significant advantages over conventional diagnostic systems with regard to assay sensitivity, selectivity, and practicality have been offered by some NPs-based assays (Alivisatos, 2003(Alivisatos, , 2004. NPs have been successfully used as labels in nucleic acid (Huber et al, 2004;Niemeyer, 2001;Wang et al, 2001). Quantum dots are also widely used due to their attractive properties to act as DNA tag (Merkoçi et al, 2005).…”

Rheological Parameters Assessment in Serum, Plasma and Whole Blood of Rats after Administration of Gold Nanoparticles of Different Sizes: In vivo

“…GNPs are used as immunostaining marker particles for electron microscopy and as chromophores for immunoreactions and nucleic acid hybridisation [12,13]. Their application for gene delivery into cells has also been reported [14][15][16][17].…”