Saturday, May 17, 2014

Spectrophotometry - Luminometry

Till now, we have seen various posts on UV-visible spectrophotometry, IR-spectrophotometry and spectrofluorimetry. Now, we will have a look at the next type of spectrophotometry which is luminometry.
As the name suggests, this type of spectrophotometry is associated with the phenomenon of luminescence. Now, the question is “what is luminescence?” Luminescence can be described as the emission of light by certain materials which do NOT result from heating (that is, the emission of light is when the temperature is below that of incandescence). Luminescence is the basic principle behind the working of luminometers. This phenomenon is usually ascribed to oxidative reactions which take place in solution producing molecules in an excited state. Some of these reactions release energy in the form of heat while others release in the form of photons.
Examples of luminescent compounds are luciferin (light emitting compound found in organisms), luminol (chemical exhibiting luminescence).

There are two major categories of luminescence as chemiluminescence and bioluminescence. It is easy to understand them as the name itself suggests the meaning. So, chemiluminescence is the luminescence produced by some chemical means. For example, luminol when oxidized with hydrogen peroxide (H2O2) in the presence of a catalyst produces luminescence which is called the chemiluminescence. On the other hand, luminescence which is produced by the interference of an enzyme is referred to as bioluminescence. 

Advantages of luminometry 
There are various advantages of luminometry over spectrophotometry. Firstly, luminometry is more sensitive as around femtomole quantities can be measured. Next advantage is that of a simple instrumentation (as we will see below). In luminometers, wavelength selectors are not required. This is so because the luminescent light is monochromatic as a result of its emission from a specific reaction.

The basic components of luminometers are:
a. A light-tight chamber in which the cuvette containing the sample can be kept

b. A facility for the addition of luminescent reagents in light-tight fashion
c. A detector (which is generally a photomultiplier)
d. An amplifier
e. A recorder

The light which is emitted by the reaction taking place in the cuvette is measured either as a peak value (which generally measures the concentration of compound of interest) or the rate of change of intensity (which is generally while measuring enzyme intensities). 


We will discuss here three main systems which are of frequent use as firefly, bacterial luminescence and luminol chemiluminescence. The principle and applications of each of these are described below:

a. Firefly luminescence and ATP measurement:

Luciferase enzyme catalyses the following reaction in the presence of magnesium:

Here, for each molecule of ATP reacting, one photon of intensity of 562nm is produced. This system is highly specific for ATP if all the reagents are pure. By linking this reaction with various other reactions, it can be used to assay a number of ATP-specific enzymes and their substrates such as creatine kinase, creatine phosphate etc.

b. Bacterial luminescence and coenzymes measurement:

The coenzymes that can be measured by this method are the NADH and NADPH. This system utilizes a purified oxidoreductase obtained from the bacterium Benecka harveyi. The reaction can be then coupled to bacterial luciferase as follows:

Here, bacterial luciferase catalyzes the oxidation of aldehyde by oxygen in the presence of FMNH2 during which a photon of maximum intensity at 495nm is produced.
c. Luminol based chemiluminescent assays:
Luminol is oxidized by hydrogen peroxide at pH 10-11 if chromium, copper or iron compounds are used as catalysts. Photons with maximum intensity at 430nm are produced.

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