Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes

BackgroundThe need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds. Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available. Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes. We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells.ResultsHeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH3)2Cl2], other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-[Pt(NH3)2Cl2] or K2[PtCl4], inhibited β-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2.ConclusionsThe EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription. Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery

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’

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

<p>Abstract</p> <p>Background</p> <p>For 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).</p> <p>Results</p> <p>Capture 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 10³TCID₅₀units for H5N1 viral RNA.</p> <p>Conclusions</p> <p>The 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.</p

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

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-dT(20) 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 μg unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps

Efficient blue emitting organic light emitting diodes based on fluorescent solution processable cyclic phosphazenes

Solution processable blue fluorescent dendrimers based on cyclic phosphazene (CP) cores incorporating amino-pyrene moieties have been prepared and used as emissive layers in organic light emitting diodes (OLEDs). These dendrimers have high glass transition temperatures, are monodisperse, have high purity via common chromatographic techniques, and form defect-free amorphous films via spin/dip coating. The solution processable blue light emitting OLEDs reach current efficiencies of 3.9 cd/A at brightness levels near 1000 cd/m2. Depending on the molecular bridge used to attach the fluorescent dendron to the inorganic core, the emission wavelength changes from 470 to 545 nm, corresponding to blue and green light respectively. Via dilution experiments we show that this shift in emission wavelength is likely associated with molecular stacking of the amino-pyrene units. © 2007 Elsevier B.V. All rights reserved