Room-Temperature Single-Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays

Abstract

We report two cDNA microarray-based applications of DNA-nanocrystal conjugates, single-nucleotide polymorphism (SNP) and multiallele detections, using a commercial scanner and two sets of nanocrystals with orthogonal emissions. We focus on SNP mutation detection in the human p53 tumor suppressor gene, which has been found to be mutated in more than 50% of the known human cancers. DNA-nanocrystal conjugates are able to detect both SNP and single-base deletion at room temperature within minutes, with true-to-false signal ratios above 10. We also demonstrate microarray-based multiallele detection, using hybridization of multicolor nanocrystals conjugated to two sequences specific for the hepatitis B and hepatitis C virus, two common viral pathogens that inflict more than 10% of the population in the developing countries worldwide. The simultaneous detection of multiple genetic markers with microarrays and DNA-nanocrystal conjugates has no precedent and suggests the possibility of detecting an even greater number of bacterial or viral pathogens simultaneously.

Quantum dots-based reverse phase protein microarray

“…The intensity of the background in the Qdot method is much higher, which might be caused by non-specific binding of the streptavidin-coated Qdot. The colloidal nature of the Qdot makes it more vulnerable to precipitation and aggregation, which we have observed previously with our DNA microarray report [8]. Several possible solutions could improve the signal-to-noise ratio in the Qdot method.…”

“…The proteins can be covalently linked to or immobilized by highcapacity absorption on a substrate surface, then detected with immunochemistry. In fact, microarrays were developed in the 1990's for genomic studies, where the massive parallel output afforded by the microarray has greatly improved the speed and scope of gene expression analysis and genotyping [6][7][8][9][10][11], to the point where DNA microarrays are now a routine analytical tool in genomics studies. By contrast, protein microarrays have not witnessed the same impressive popularity, mainly because the adsorption chemistry of the proteins onto the surface poses many challenges, and no convenient detection mechanism has yet become firmly established [5].…”

“…Recent progress in synthesis has allowed the highly efficient conjugation of the CdSe/ZnS nanocrystals to a wide variety of biomolecules such as DNA [21,22], proteins [23], antibodies [24,25], or short peptides [26]. Several years ago, we successfully adapted the Qdot technology for the study of cDNA microarrays [8]. By devising a highly efficient means to achieve Qdot-DNA linkage, we showed that single nucleotide polymorphism detection of genomic DNA could be reached within minutes, at room temperature, with true-to-false signal ratios above 10[8].…”

Size and Shape Effect on Biomedical Applications of Nanomaterials

“…Having already shown considerable promise as intracellular imaging and tracking agents [68] quantum dots made of CdSe and ZnS, with few exceptions [69] have not been widely investigated as materials for biodetection assays. The first example of chemically modifying CdSe quantum dots with DNA involved ligand exchange coupled with particle surface engineering [70].…”

Semiconductor nanocrystals for biological imaging

“…With continuous efforts in developing high quality biocompatible QDs, nanoparticles conjugated to antibodies, peptides and DNA have been prepared and targeted to cells and tissues specifically, allowing multiplexed labeling and long term studies that can not be achieved by using standard dyes [22][23][24][25][26][27][28][29]. Although QDs and organic dyes can have comparable quantum yields, the larger absorption cross-section of the nanocrystal results in a much stronger photoluminescence signal.…”