James J. Storhoff

SNP identification in unamplified human genomic DNA with gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the ‘point of care’.

Nanoparticle-Based biobarcode amplification assay (BCA) for sensitive and early detection of human immunodeficiency type 1 capsid (p24) antigen.

Nanotechnology-based techniques are being widely evaluated in medical testing and could provide a new generation of diagnostic assays due to their high degrees of sensitivity, high specificity, multiplexing capabilities, and ability to operate without enzymes. In this article, we have modified a nanoparticle-based biobarcode amplification (BCA) assay for early and sensitive detection of HIV-1 capsid (p24) antigen by using antip24 antibody-coated microplates to capture viral antigen (p24) and streptavidin-coated nanoparticle-based biobarcode DNAs for signal amplification, followed by detection using a chip-based scanometric method. The modified BCA assay exhibited a linear dose-dependent pattern within the detection range of 0.1 to 500 pg/ml and was approximately 150-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA). No false positive results were observed in 30 HIV-1-negative samples, while all 45 HIV-1 RNA positive samples were found HIV-1 p24 antigen positive by the BCA assay. In addition, the BCA assay detected HIV-1 infection 3 days earlier than ELISA in seroconversion samples. Preliminary evaluation based on testing a small number of samples indicates that the HIV-1 p24 antigen BCA may provide a new tool for sensitive and early detection of HIV-1 p24 antigen in settings where HIV-1 RNA testing is currently not routinely performed.

Strategies for Organizing Nanoparticles into Aggregate Structures and Functional Materials

In this review, we focus on some of the most successful approaches to date for organizing nanoparticles into macroscopic aggregates and functional materials. The preparation and resulting properties of two- and three-dimensional arrays of nanoparticles are detailed, and some potential uses for these materials are discussed. Although many types of nanoparticles can be organized into such structures, this review focuses specifically on Au and CdE (E=S, Se) nanoparticles. Gold nanoparticles are easily prepared, highly stable, well-studied, and excellent models for other metal colloids. CdE colloids are the most extensively studied semiconductor nanoparticles and hold much promise in the optoelectronics field.

Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

BackgroundFor more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP)-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2).ResultsCapture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M) gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA) and neuraminidase (NA) genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD) of the assay was less than 100 fM for purified PCR fragments and 103 TCID50 units for H5N1 viral RNA.ConclusionsThe NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2). The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.

Chemistry of Oligonucleotide-Gold Nanoparticle Conjugates

Conjugates prepared by immobilizing thiol-terminated oligonucleotides onto gold nanoparticles form stable colloidal solutions in aqueous media. The oligonucleotides can serve as linkers to organize the gold particles reversibly into three dimensional assemblies, and the gold particles can function as colorimetric reporters for hybridization of the bound oligomers with target oligonucleotides in solution.

Diagnostic Detection Systems Based on Gold Nanoparticle Probes

The development of a nanoparticle based detection methodology for DNA microarray applications is described. Originally reported by Mirkin and co-workers, the technology utilizes gold nanoparticles derivatized with thiol-modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized DNA capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. For maximum signal strength a layer of silver is deposited onto the gold nanoparticles, providing for highly sensitive and specific detection of target sequences using low cost optical detection systems. The relative optical detection limits for silver amplified gold nanoparticle probes and Cy3 based fluorescence have been tested. Furthermore, we report progress towards the direct detection of non-amplified genomic DNA from infectious agents and single nucleotide polymorphisms (SNP) in human genomic DNA based on nanoparticle technology.