Vidya Srinivasan

M.S.

2005-2007

CV

srini46@.msu.edu

Cost effective detection of multiple waterborne pathogens with gold nanoparticles and silver enhancement on a microarray using a flatbed scanner

High throughput detection of pathogens is of interest for numerous applications including biological quality assessment of water. Microarray technology is the most promising platform due to its capability for multiplexing. Conventional approaches for microarray based detection employ prohibitively expensive protocols using flourophore and laser scanner based detection. There exists a need for the development and validation of more economical alternatives. In this area of research, we exploit the recent advances in nanoparticle based microarray technology for the detection of multiple waterborne pathogens. An in-house database containing 3,183 sequences (virulence and marker genes related to pathogens) was used for the probe design. The sequences were obtained from GenBank. The database in total, contains 42 genera, 97 species, and 509 genes. Oligonucleotide probes were designed using a software called CommOligo, (He, Z., L. Wu, X. Li, M. W. Fields, and J. Zhou. 2005. Empirical establishment of oligonucleotide probe design criteria. Appl. Environ. Microbiol. 71:3753-3760). A total of 10,802 probes (50 mer) were designed.
Forty seven virulence and marker genes from twelve pathogens including Campylobacter jejuni, Staphylococcus aureus, and Vibrio cholerae were targeted for the initial set of experiments. A silver enhanced gold nanoparticle based signal generation strategy was optimized. An extensive set of oligonucleotides for selected pathogens were synthesized in situ on glass slides (in quadruplicate) using the technology developed by Prof. Erdogan Gulari’s group (Department of Chemical Engineering, University of Michigan). Imaging of the microarrays was performed using a flatbed scanner (2 µm resolution, compared to 5 µm resolution for a conventional laser scanner). Fig 1 (a) and Fig 1 (b) are scanned images of 2 hybridized glass arrays using the flatbed scanner at a resolution of 2 µm.

Figure 1. The middle panel shows scanned images of the hybridized glass array using a flatbed scanner at 2 µm resolution. On the left is scanned chip using laser scanner. Right panel shows that for larger spots, no scanning is necessary.

Target DNA was labeled with biotin, and gold nanoparticles were introduced as streptavidin conjugates. Signal amplification was subsequently achieved using silver-enhancement. High specificity was achieved through optimization of the hybridization conditions, and increased redundancy in the probe sets. Optimal signal to noise ratios were obtained after 15 min of silver enhancement. Replicate hybridizations of known target samples, including hybridizations of unknown target samples were carried out.