AmpliPHOX

Ampliphox (stylized "ampliPHOX") is a colorimetric detection technology created as a research tool for the analysis of low-density microarrays using colorimetric detection. It combines a reagent kit and a benchtop instrument for detection for low-density (<2500 spots) microarray applications and is produced by InDevR, Inc. The ampliphox provides equivalent analytical sensitivity to fluorescence by using colorimetric readout.

The process underlying the ampliphox colorimetric detection technology is a light-starting chemical reaction that forms solid polymer spots on the microarray. Biotinylated targets are first captured onto the microarray and subsequently labeled with a streptavidin-coupled initiator. When a proprietary solution is added and light from the ampliphox reader illuminates the array, polymer grows selectively from only locations where the biotinylated targets were captured. The process takes a couple of minutes, and can be seen with the unaided eye and subsequently imaged with the ampliphox reader.

Principles of operation

The ampliphox assay allows for the detection of biotin-labeled targets using light and a proprietary streptavidin-label to start the polymerization of a highly optimized monomer solution (amplify). Only areas where a marking event has taken place produce transparent polymer. The contrast of the polymer is enhanced by applying a simple stain prior to imaging and analysis using the ampliVIEW software. Polymer spots formed are visible to the unaided eye.

Biotin molecules must be incorporated into the microarray target(s) before ampliphox detection is performed. This can be achieved using several commercially available or custom options, depending on the particular application. Brief descriptions of the key steps and features of the ampliphox assay and ampliPHOX Reader are given below.

Applications

Ampliphox has been optimized for use with glass microarray substrates and can be used with both nucleic acid and protein-based systems, and, in principle, can detect any biotin-labeled product. Research at the USDA has used this technology to profile Shiga toxin-producing Escherichia coli by identifying the O-antigen gene clusters and virulence genes. Research at InDevR was performed to identify and type influenza by using ampliphoxX and InDevR's custom microarrays with RT-PCR. This work shows that ampliphox detection paired with a low-density microarray can provide a low-cost alternative to methods such as QRT-PCR for surveillance of influenza, particularly in resource-limited settings.