Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays.

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.

[1]  A Chakravarti,et al.  High-throughput variation detection and genotyping using microarrays. , 2001, Genome research.

[2]  John E. Johnson,et al.  Fluorescent signal amplification of carbocyanine dyes using engineered viral nanoparticles. , 2006, Journal of the American Chemical Society.

[3]  W. Tan,et al.  Using luminescent nanoparticles as staining probes for Affymetrix GeneChips. , 2007, Bioconjugate chemistry.

[4]  Olivier Ferraris,et al.  Use of the DNA Flow-Thru Chip, a Three-Dimensional Biochip, for Typing and Subtyping of Influenza Viruses , 2004, Journal of Clinical Microbiology.

[5]  O. Cussenot,et al.  DNA microarrays in clinical practice: past, present, and future , 2003, Clinical and Experimental Medicine.

[6]  Banahalli R Ratna,et al.  Toward single molecule detection of staphylococcal enterotoxin B: mobile sandwich immunoassay on gliding microtubules. , 2008, Analytical chemistry.

[7]  David A Stenger,et al.  Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays. , 2006, Genome research.

[8]  D. Stenger,et al.  Nucleic Acid Amplification Strategies for DNA Microarray-Based Pathogen Detection , 2004, Applied and Environmental Microbiology.

[9]  J. Derisi,et al.  Microarray-based detection and genotyping of viral pathogens , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[10]  John E. Johnson,et al.  New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus. , 2004, Chemistry & biology.

[11]  John E. Johnson,et al.  A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer. , 2006, Biosensors & bioelectronics.

[12]  M. Bunster,et al.  Crystallization and 2.2 A resolution structure of R-phycoerythrin from Gracilaria chilensis: a case of perfect hemihedral twinning. , 2001, Acta crystallographica. Section D, Biological crystallography.

[13]  Moon J. Kim,et al.  Separation and recovery of intact gold‐virus complex by agarose electrophoresis and electroelution: Application to the purification of cowpea mosaic virus and colloidal gold complex , 2004, Electrophoresis.

[14]  Frances S. Ligler,et al.  Application of Broad-Spectrum, Sequence-Based Pathogen Identification in an Urban Population , 2007, PloS one.

[15]  M. Kronick,et al.  The use of phycobiliproteins as fluorescent labels in immunoassay. , 1986, Journal of immunological methods.

[16]  James M. Eldred,et al.  Viral Discovery and Sequence Recovery Using DNA Microarrays , 2003, PLoS biology.

[17]  M. Bittner,et al.  Expression profiling using cDNA microarrays , 1999, Nature Genetics.

[18]  I-Ming Hsing,et al.  Enhanced electrochemical detection of DNA hybridization based on electrode-surface modification , 2003 .

[19]  Gary J. Vora,et al.  Identifying Influenza Viruses with Resequencing Microarrays , 2006, Emerging infectious diseases.

[20]  Weihong Tan,et al.  Development of Organic‐Dye‐Doped Silica Nanoparticles in a Reverse Microemulsion , 2004 .

[21]  Douglas R. Call,et al.  Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of Listeria monocytogenes from Genomic DNA , 2005, Journal of Clinical Microbiology.

[22]  J. Hacia.  Resequencing and mutational analysis using oligonucleotide microarrays , 1999, Nature Genetics.

[23]  Weihong Tan,et al.  Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles. , 2003, Journal of the American Chemical Society.