Chemiluminescence is the generation of electromagnetic radiation as light by the release of energy from a chemical reaction. While the light can, in principle, be emitted in the ultraviolet, visible or infrared region, the reactions emitting visible light are the most common. They are also the most interesting and useful.
In order to achieve the highest levels of sensitivity, a chemiluminescent reaction must be as efficient as possible in generating photons of light. Each chemiluminescent compound or group can produce no more than one photon of light. A perfectly efficient reaction would have a chemiluminescence quantum yield ΦCL of one, i.e. one photon/molecule reacted according to the equation:
ΦCL = ΦCE × ΦF × ΦR
The chemiexcitation quantum yield ΦCE is the probability of generating an electronic excited state in a reaction and has a value between 0 and 1, with 0 being a completely dark reaction and, when 1, all product molecules are generated in the excited state. The most useful chemiluminescent reactions will have a ΦCE of about 10-3 or greater.
One reason accounting for the growing popularity of chemiluminescent assays is their exquisite detection sensitivity. Unlike absorbance (colorimetric) or fluorescent measurements, assay samples typically contribute little or no native background chemiluminescence. Measurement of light intensity is relatively simple, requiring only a photomultiplier or photodiode and the associated electronics to convert and record signals. The lack of inherent background and the ability to easily measure very low and very high light intensities with simple instrumentation provide a large potential dynamic range of measurement.
Factors in Choosing a Detection Reaction:
Enzyme Label or Direct Label?
A common means for generating chemiluminescence is to use a label enzyme to catalyze a chemiluminescent reaction. Each label can initiate multiple chemiluminescent reactions, so it is usually only necessary to incorporate one or a few enzyme labels/analyte. The chemiluminescent compound is supplied as the enzyme substrate in excess to assure saturation kinetics. The lifetime of the excited state product P* → P (step 5) is extremely short in comparison to the other steps and has no effect on the observed reaction kinetics.
The chemiluminescence intensity/time profile consists of an initial rise period up to a prolonged emission at a plateau or pseudo-plateau level. Light intensity at any time point through the plateau can be related to the amount of enzyme. Measurement can be made during the rising portion if speed is an issue as either a single point or a multi-point slope type measurement. For maximal sensitivity, measurement can be performed on the plateau.
Factors in Choosing Assay Format:
Homogeneous or Heterogeneous?
It is far simpler to design heterogeneous assays in which labeled binding pair complexes are separated from unbound labeled reactants. Most chemiluminescent reactions can be adapted to this assay format, by labeling either with a chemiluminescent compound or with an enzyme and using a chemiluminescent substrate. Most commercially developed immunoassays are of this type.
The attractiveness of chemiluminescence as an analytical tool is the simplicity of detection. The fact that a chemiluminescent process is, by definition, its own light source means that assay methods and the instruments used to perform them need only provide a way to detect light and record the result. Luminometers need consist of only a light-tight sample housing and some type of photodetector. Taken to the extremes of simplicity, photographic or x-ray film or even visual detection can be used.
The simple requirements of chemiluminescent methods make them robust and easy to use. But what about sensitivity?
Chemiluminescence has two built-in advantages here, too.
Most samples have no 'background' signal, i.e. they do not themselves emit light. No interfering signal limits sensitivity.
Measurement of chemiluminescence is not a ratio measurement in the way fluorescence and absorption or color are. In fluorescence this can lead to difficulties with fluorescers with a small Stokes shift. Fluorescence may not be easy to resolve from the exciting wavelength.
Photomultiplier tubes (PMTs) have traditionally been the workhorse detector in luminometers. Their advantages include good sensitivity, a broad dynamic range and applicability over a reasonably broad spectral range. PMTs are known for their very low dark currents leading to excellent signal to noise for low intensity samples.
PMT based systems operate in two basic modes, single photon counting and current sensing. There are examples of hybrid systems which are single photon counting to a light level in the low millions of photons/second and then switch to current sensing above that level.
PMT single photon counting systems are capable of exquisite sensitivity. Use of this type of detector is the method of choice for low light detection and quantitation as in, for example, detecting the ultraweak luminescence associated with phagocytosis. The greater sensitivity comes at a cost however. Sample housings must be very light-tight. Moderate light levels saturate the detector; high levels can cause irreversible damage to the PMT.
PMT current sensing systems are also capable of excellent sensitivity and will often read higher light levels than single photon counting systems without damage.