Tuesday, July 3, 2012

Fluorescent versus Chemiluminescent Detection

When I first started Arrayit in 1996, nucleic acids, proteins, cells, or tissues were commonly labeled with radioactive labels, and images for analysis was captured on film in macroarrays and plate based formats.  The first microarray publication really highlighted the value of fluorescent detection and miniaturization.  Safety concerns, convenience, ease of use and sensitivity, spurred the development of alternative detection techniques, and today, researchers can choose from a range of options, including fluorescence, colorimetric and chemiluminescence, and in some cases radiography is still used.  Label free detection technologies are also much more available, but I'm not going to write about those today.

Fluorescence occurs when light is absorbed from an external excitation source by a fluorescent molecule called a fluorophore and subsequently emitted. The period of excitation and emission lasts until the excitation source is turned off (typically a laser) or the fluorophore is consumed (photo bleached or dead due to some other chemical reaction). A fluorescent probe is any small molecule that that undergoes changes in one or more of its fluorescent properties as a result of noncovalent interaction with a protein or other macromolecular structure. There are many different fluorescent molecules, both naturally occurring and synthetic, each molecule having distinctive spectroscopic properties. This variety of molecules is exploited to enable highly localized, sensitive detection of chemical reactions, for tagging and identification of molecular structures, for monitoring of cellular states, and even for monitoring multiple properties simultaneously. Due to their ability to multiplex, higher sensitivity than chromogenic dyes, fluorescent probes can be used to effectively signal the presence of minute amounts of a specific protein, DNA or RNA without the hazards associated with radioactive labels.

Figure 1. Stoke shift of the standard microarray dyes are far enough part so that the two channels (red and green) do not overlap and "cross talk" allowing 2-color microarray experiments to be performed.

Chemiluminescence is the production of visible light (luminescence) occurring as a result of a chemical reaction. It is most often used with an enzyme label called horseradish peroxidase and a chemiluminescent substrate (example Luminol). Chemiluminescence can be more sensitive than other detection techniques due to the amplification of signal by the enzymes.  However, the signal is not stable, it lasts for 6-24 hour depending on the substrate. How much light is generated and for how long depends on the specific substrate being used and the enzyme-to-substrate ratio present in the system. Although the amount of substrate in any assay can be relatively constant, the amount of enzyme present depends on how much was added and other factors. Too much enzyme conjugate applied to the assays is the single greatest cause of signal variability, background noise, short signal duration and low sensitivity.  A signal emission dies out slowly is most desirable as it demonstrates that each component of the system has been optimized and allows reproducible results. This characteristic is much easier to achieve with fluorescent detection reagents.  A signal that decays too quickly can cause variability, low sensitivity and lack of signal detection and therefore loss of quantitation.  A long-lasting signal minimizes variability with transfer efficiency, different manufacturer lots of detection reagents and other factors. Although HRP continues its activity for as long as substrate is available, a predictable statistical probability exists for various substrates to catalyze HRP and render it inactive. Free radical production during the oxidation reaction can bind to HRP in such a way to at best make it plateau in signal for a few hours.

Arrayit's preferred method for detection is fluorescent.  It offers equivalent, if not higher, signal to noise ration than isotope and chemiluminescent detection systems and have the added advantage of greater versatility, multiplexing, stability, ease of use and safe handling.  Further, genetic and proteomic labeled probes give us the platform for making microarrays to use as biosensors for all types of quantitative assays including gene expression, aCGH, genotyping, immunoassays, and more...  With the labels giving us the ability to measure proper positive and negative controls in 2 color multiplexed assays. For more information email me at todd@arrayit.com and visit our website, www.arrayit.com