Photopolymerization as an Innovative Detection Technique for Low-Density Microarrays

Abstract

One limitation that accounts in part for the scarcity of commercially available diagnostic microarrays is the expense associated with fluorescence detection. Here we present a colorimetric method based on photopolymerization as an "on-chip" signal amplification technique. Proof of principle experiments are detailed and followed by the use of a simple influenza microarray to demonstrate the technique for the first time with clinical samples. The advantages of this new technique include rapid (<5 min) signal amplification ( approximately 105) in ambient conditions for both DNA and protein microarrays, low reagent cost (<$1 per assay), visual or inexpensive detection, and signal preservation for at least two years under ambient conditions.

Identification ofEscherichia coliO157 by Using a Novel Colorimetric Detection Method with DNA Microarrays

“…To implement the use of improved detection methods with DNA microarrays, the present study evaluated photopolymerization, a novel colorimetric detection method (Kuck and Taylor, 2008;Sikes et al, 2008;Dawson et al, 2009), for genotyping E. coli O157 strains. To provide a proof of concept for testing photopolymerization, a low-density 30-mer DNA oligonucleotide array was constructed to target the E. coli O157 virulence genes eae, per, stx1, and stx2 (Table 1).…”

“…The specific colorimetric detection of signals on the microarray were detected with the ampliPHOXÔ Detection System (InDevR, Inc.), a light-initiated signal amplification through polymerization (Kuck and Taylor, 2008;Sikes et al, 2008;Dawson et al, 2009). The hybridized microarrays were labeled subsequently with ampliTAGÔ labeling solution (InDevR, Inc.) for 5 min at room temperature in a dark humidity chamber, and immediately after labeling, microarrays were rinsed briefly with Wash Buffer D, Wash Buffer C for 5 min, and then dried.…”

“…One challenge of using the DNA microarray platforms for pathogen detection has been developing cost effective as well as sensitive procedures that would indicate the positive signals on the array (Call, 2005;Kuck and Taylor, 2008;Vora et al, 2008). Fluorescent assays are commonly used methods for microarray-based detection of pathogens.…”

“…Fluorescent assays are commonly used methods for microarray-based detection of pathogens. However, limitations of these assays are that labeling of target DNA can be inconsistent and highly variable, and they utilize expensive and nonportable scanners for data acquisition and analysis (Call, 2005;Kuck and Taylor, 2008;Vora et al, 2008). Other nonfluorescent, colorimetric assays employ the use of unstable reagents that require temperature-controlled environments and that have variable development times, leading to an increase in nonspecific backgrounds (Kuck and Taylor, 2008).…”

“…Improved procedures for pathogen surveillance are thus needed with sufficient sensitivity, cost effectiveness, and suitability for routine testing. To implement the use of better detection methods for categorizing E. coli O157 strains, the present study evaluated ampliPHOX, a novel and innovative colorimetric procedure that is based on light-initiated signal amplification through polymerization (Kuck and Taylor, 2008;Sikes et al, 2008;Dawson et al, 2009). Our findings demonstrate that photopolymerization is a simple and rapid method for the accurate genotyping of E. coli O157 strains with DNA microarrays.…”