What is chemiluminescence?

Chemiluminescence is the conversion of chemical energy to light energy. Energy produced during the chemical reaction is released as light energy as the reaction products decompose and decay from a high energy state to a lower energy state. Chemiluminescence provides the highest detection sensitivity compared to fluorescent or colorimetric detection methods.

1,2-dioxetane chemiluminescent substrates

1,2-Dioxetane chemiluminescent substrates enable highly sensitive detection of biomolecules in both solution-based tube or microplate assays as well as membrane-based detection assays. Light emission is detected using luminometer platforms, including singlemode and multi-mode instrumentation, incorporating either photomultiplier tube or photodiode detectors, CCD camera, X-ray, or photographic film imaging. We have commercially available substrates for alkaline phosphatase, b-galactosidase, and other enzymes. Figure 1 shows the mechanism of light emission of the Tropix CDP-Star substrate. Chemiluminescent products are available as substrate concentrates or ready-to-use solutions with or without proprietary Tropix polymeric chemiluminescence enhancers.

Figure 1. Light Emission Mechanism of CDP-Star Substrate.

Substrate/enhancer formulations

Chemiluminescence enhancers provide a significant increase in light emission efficiency with 1,2-dioxetanes and are essential for use with solution-based assays.

CSPD and CDP-Star substrates are available as ready-to-use solutions including one of two polymeric enhancers: Sapphire-II enhancer and Emerald-II enhancer. Sapphire-II enhancer typically provides slightly higher signal-to-noise ratios than Emerald-II; while Emerald-II enhancer provides the highest signal intensity. The choice of ancoptimal substrate/enhancer formulation depends on the instrumentation being used (spectral sensitivity), the type of microplate being used, and the desired signal intensity. The detection sensitivity obtained with the various substrate/enhancer formulations is very similar and a dynamic range of alkaline phosphatase quantitation of 5–6 decades of enzyme concentration is achieved with all formulations.

“Glow” light emission kinetics

Tropix 1,2-dioxetane alkaline phosphatase substrates provide “glow” light emission that reaches peak light signal intensity quickly and is sustained over many hours. This provides great flexibility in assay measurement time and minimal signal intensity variation across a microplate. The glow light emission kinetics achieved with these reagents allow great flexibility in the choice of assay temperature, measurement interval, and assay detection instrumentation.

Immunoassay performance

The substrate/enhancer formulations also provide both superior sensitivity and kinetic performance compared to numerous HRP/luminol-based detection systems. A sandwich immunoassay quantitating recombinant human IL– 6 incorporates a biotinylated detector antibody and either streptavidin-AP or streptavidin-HRP followed by the respective chemiluminescent detection chemistries. With CSPD or Sapphire-II ready-to-use substrate/enhancer formulation, detection sensitivity of <0.5 pg/mL is achieved (~10–50-fold better than HRP/luminol detection sensitivity) with a dynamic range of detection of four orders of magnitude of IL–6 concentration.

With CSPD or Sapphire-II ready-to-use, glow light emission kinetics are long-lived and constant light intensity signal enables measurement at 37°C if desired. In contrast, the light emission obtained with HRP/luminol-based detection chemistries typically demonstrates rapid decay after a nearly immediate light emission maximum.

Custom formulations

We'll be your partner in the development of custom formulations of existing and new chemistries to provide chemiluminescent detection solutions that meet your specific assay and instrumentation needs. Contact our experts to learn more about using chemiluminescence solutions in your research applications or to inquire about OEM and custom chemiluminescence solutions for your high-performing applications and kits.

outlicensing@thermofisher.com