AmpliPHOX

The ampliPHOX Colorimetric Detection Technology was created as a research tool for the analysis of low-density microarrays by providing rapid, cost effective colorimetric detection with minimal user input. The combination of a simple reagent kit and a small benchtop instrument provide a powerful new detection technology for low density (<2500 spots) microarray applications and is produced by InDevR, Inc. With an investment about ten times lower than that required for a typical microarray scanner, ampliPHOX provides equivalent analytical sensitivity to fluorescence with a simple colorimetric readout.

The key to ampliPHOX Colorimetric Detection Technology is a light-initiated 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 occurs in just a few 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 (ampliTAG) to initiate the polymerization of a highly optimized monomer solution (ampliPHY). Transparent polymer is formed only on spots where a labeling event has occurred. The contrast of the polymer is enhanced by applying a simple stain (ampliRED) prior to imaging and analysis using the ampliVIEW software. Polymer spots formed are visible to the unaided eye. On the InDevR website, a video is available to illustrate the concept.

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

Labeling

The incorporation of ampliTAG onto the biotinylated microarray is the first step toward detecting the microarray capture event. Labeling the microarray with ampliTAG is performed manually and is not accomplished using the ampliPHOX Reader. The microarray labeling process consists of a five-minute incubation step followed by a five-minute wash.

Signal Amplification

The ampliPHY solution is applied manually to the labeled microarray and exposed to light using the instrument’s Photoactivation Bay. During photoactivation, the ampliTAG triggers polymerization of the ampliPHY. The result, after excess ampliPHY is removed, is a colorless yet visible polymer present only in regions where targets were labeled with ampliTAG. This polymer is subsequently stained with ampliRED to make the spots visible to the unaided eye and to allow for simple digital imaging and analysis of resulting signals using the ampliPHOX Reader and accompanying ampliVIEW software.

Data Analysis

After the polymer has been stained, the slide is transferred to the instrument’s Imaging Bay for analysis. First, a simple digital image of the array is captured. This image is compiled with other important information about the microarray into a data file with a “.ari” extension. Once saved, .ari files can be analyzed at any time using the Analysis Tab within the ampliVIEW software. A target is considered positive if all spots for the given target meet the following criteria:

${\bar {x}}$_s > ${\bar {x}}$_b + 1.8 σ_b

Where ${\bar {x}}$_s is the mean spot intensity, ${\bar {x}}$_b is the mean background intensity (calculated from 48 pixels), and σ_b is the standard deviation of the background intensity.

Applications

ampliPHOX has been optimized for use with glass microarray substrates and can be utilized with both nucleic acid and protein-based systems, and in principle can be used to detect any biotin-labeled product. Research at the USDA has utilized this technology to profile Shiga toxin-producing Escherichia coli by identifying the O-antigen gene clusters and virulence genes. Additionally, research at InDevR was performed to identify and type Influenza by using ampliPHOX and InDevR's Custom Microarrays in conjunction with RT-PCR. This work demonstrates 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.