Introduction

The Potential of Microarray Platforms

With the advent of rapid genome sequencing and large genome databases, it is now possible to take advantage of genetic information in a myriad of ways. One of the most promising technologies is microarrays that use DNA for selective capture of genomic targets (1). DNA microarrays, also known as biochips, have found widespread use in gene expression analysis, and there are now several academic demonstrations of biochips used in diagnostics (2). An exciting aspect of biochip platforms is the capability to screen for multiple pathogens simultaneously. DeRisi and co-workers demonstrated a “virus chip” that contained sequences for hundreds of viruses, including many that cause respiratory illness (3). In the DeRisi work, the genetic material derived from clinical samples was amplified by polymerase chain reaction (PCR) prior to capture, and fluorescent labels were used for detection. Recently, Rowlen and co-workers at the University of Colorado and the Centers for Disease Control in Atlanta developed a microarray (FluChip) for rapid strain analysis of influenza (4,5,6,7). The FluChip also relied on PCR amplification of targets and fluorescence detection on the array. While DNA microarrays are in some respects ideal for surveillance applications, there are several practical issues that currently prevent their widespread use as diagnostic tools. One such limitation is the use of fluorescence detection, which necessitates the use of expensive, high quality, nonfluorescent substrates, stringent handling conditions, and expensive (>$10K U.S.), non–field portable fluorescence microarray readers. Effective global monitoring of biological pathogens will require simple, robust, and inexpensive detection methods and instrumentation.

Current Colorimetric Methods for Microarrays

There are two commonly used colorimetric methods for signal amplification or detection on microarrays: one based on silver staining or silver nanoparticles (8) and the other based on a colored precipitate that results from alkaline phosphatase (AP) action on a substrate (9). The alkaline phosphatase reaction typically involves the binding of AP-conjugated streptavidin to biotin-labeled targets on the microarray surface. Addition of a substrate results in a colored, often blue, precipitate formed only where the enzyme is bound to the target. The sensitivity of this system has been demonstrated to be comparable to that of fluorescence detection (10) and the advantages include visual or inexpensive detection and inexpensive reagent costs (∼$2 per assay). The disadvantages of the AP detection method include unstable reagents that must be stored frozen until use and a variable development time that can lead to “overexposure” and higher nonspecific backgrounds (Arrayit AP kit [Telechem Corporation, Sunnyvale, CA, USA] and protocol: www.arrayit.com/Products/Labeling_Kits/APK/apk.html).

An excellent example of the silver nanoparticle method is the Eppendorf Silverquant kit and associated protocol (Eppendorf [Hamburg, Germany] and protocol: www.eppendorfna.com).The method involves labeling biotinylated DNA targets on the microarray with streptavidin-conjugated colloidal gold, which subsequently serves as a reduction surface for silver ion. The result is a gray silver precipitate that can be visualized by eye or quantified by either transmission (8) or resonance light scattering with sensitivity comparable to fluorescence. The advantages of the silver precipitation method include colorimetric detection and signal stability. Disadvantages include cost per assay (∼$6 U.S.) and unstable reagents that must be refrigerated and used in a temperature-controlled environment.

Photopolymerization as a Signal Amplification Method

An innovative approach to detection on microarrays, which eliminates the need for fluorescence reagents and expensive scanners, is based on photopolymerization of an organic monomer. The concept of the non-enzymatic signal amplification (NESA) system is shown schematically in Figure 1. Detection is based on the use of a photoinitiator as the label rather than a fluor. Once the labeled molecule is captured, the resulting complex is covered in a solution containing monomers and irradiated at a wavelength absorbed by the photoinitiator. Light absorbed by the photoinitiator produces free radicals that propagate by radical addition between the surrounding monomers thereby forming a polymer (much like the photochemical process used for UV-curable dental fillings). For a low-density microarray the process can be completed in minutes. The result is a solid polymer located exclusively where the target and photoinitiator are bound to the substrate. Once excess monomer is washed away, the polymer can be visualized by eye.