What does chemiluminescence mean?


The word ‘chemi’ indicates that it has something to do with chemicals and the word ‘luminescence’ means that it gives off light. The combination of the two words- chemiluminescence means ‘generating light via a chemical reaction.’ To get a better understanding of how chemiluminescence works here is an example. When you turn on an incandescent light bulb, an electric current passes through the filament. As there is always some resistance to the flow of current, the metal filament starts to get hot. This results in excitation of the electrons or a higher state of energy. When the electrons relax or return to their ground state, all the heat energy is then released as light.


There are many types of chemical reactions where the energy is emitted not as heat, but light. These reactions are known as chemiluminescent reactions and also occur in living organisms like the jellyfish. However, the most well-known land creature that generates light is the firefly. In this insect, there is an enzyme called luciferase (meaning light-bearing) which triggers a reaction that generates energy emitted as light- which can be seen in the insect’s lower abdomen as a flashing beacon.


Besides the firefly, chemiluminescence can also be seen in earthworms, some fungi and marine life. Many marine species living at great depths of the ocean rely on light. These deep sea organisms have evolved to produce light to attract and catch other prey. Sometimes the light is used to attract others creatures of the same species. A variety of microorganisms also use bioluminescence to communicate with each other. The most common colors emitted by marine life and bacteria are green and blue, because they tend to have a short wavelength compared to red. In addition, blue and green colors can be transmitted more easily in water. The same chemiluminescent process is also seen in light sticks that are sold to hitchhikers, campers and sports fans. Over the years, scientists have exploited the light emitting reactions to develop a number of laboratory tests.


What is the difference between Fluorescence, Phosphorescence, and Chemiluminescence?


In the world of luminescence chemistry, there are several types of ‘light’ reactions which most people think are all the same but in reality are slightly different from each other. People who work in a laboratory should know the difference between fluorescence, phosphorescence, and chemiluminescence


Fluorescence is basically the emission of light by an object that has absorbed either electromagnetic radiation or some type of light. The object then emits this light. In the majority of cases, the emitted light has a longer wavelength and therefore a lowered state of energy than the absorbed light/radiation. A classic example of fluorescence is known to occur when the absorbed radiation or light is in the ultraviolet region of the color spectrum, and thus the human eye cannot see the light. But when the light is emitted, it is in the visible spectrum. This gives the fluorescent compound a specific color that can only be seen when it is exposed to ultraviolet light.


In general, fluorescent items will stop glowing almost immediately when the radiation source ceases. On the other hand, phosphorescent materials will continue to emit light for a lot longer period even when the radiation exposure has stopped.


The use of fluorescence has seen applications in many industries including medicine mineralogy, laboratory (fluorescence spectroscopy), use of fluorescent labeling, biological detectors, dyes and even fluorescent lamps. The process of fluorescence is also known to occur in some minerals and in several organisms of the animal kingdom.


Phosphorescence is a type of photoluminescence and closely related to fluorescence. However, unlike fluorescence, a compound that is phosphorescent will not immediately re-emit the radiation that it has absorbed. The reason for the delay in remitting is due to the ‘forbidden’ energy state transitions in quantum physics. Because the transition in energy occur gradually in certain compounds, the absorbed radiation will only emit light at a low intensity for several hours after the original excitation. Common examples of phosphorescent materials include items that glow in the dark such as certain paint, stickers, and dials on clocks that glow after they have been charged with the bright light. In most cases, the glow starts to slowly fade out, sometimes in a matter of minutes or may last a few hours in a dark room. It was this observation of phosphorescent materials that initially led to the discovery of radioactivity more than a century ago.


Chemiluminescence is a term for light that is generated as a product of a chemical reaction. The chemiluminescent reactions generate unstable compounds, which then break down or decay in order to form more stable compounds; in the process, energy is emitted in the form of light, which is visible.


Where is Chemiluminescence used outside the laboratory?


Outside of laboratories, chemiluminescence is also used to make several products of which the most well known are light sticks. These glow sticks are widely used by emergency workers, police, military personnel, Halloween tricksters and road workers. The amount of light generated by the light sticks is not intense but they are ideal for highlighting hazards on the road or identifying a person in the dark. Light sticks are often used during night time scuba diving, following natural disasters, when there has been a traffic accident at night or to mark a pathway. The other benefit of light sticks is that they can be used when electricity may not be available or be dangerous, such as after a natural disaster. To turn on the light in the sticks, the middle segment is snapped which initiates a chemical reaction. The resulting reaction results in light of bright colors. Most nightsticks contain hydrogen peroxide and Cyalume, (A phenyl oxalate ester) which are kept separate until the stick is snapped. When the two chemicals react, they generate hydrogen peroxide and an unstable peroxy compound. As the unstable peroxy compound become stable, it combines with a dye, which results in a glow.


cyalume (phenyl oxalate ester) + H2O2 + dye → phenol + unstable cyclic peroxy compound + dye


→ phenol + 2CO2 + dye (excited)


What is a chemiluminescent immunoassay?


A chemiluminescent immunoassay is a slight modification of the well-known enzyme immunoassay (EIA), which is a widely used biochemical technique in microbiology and immunology. During an enzyme immunoassay, the technique utilizes enzyme-labeled antibodies to detect the presence of antigens in body fluids, usually serum, urine or CSF. This test is widely used to make the diagnosis of many immunological disorders. The antigen is first attached to a surface and then the antibody with an enzyme is added to the substrate. The subsequent reaction between the antigen and antibody produces a chemical reaction, which is often detected as a change in color of the substrate or a visible signal. The intensity of light generated is then measured using spectrophotometry. The sensitivity of the detection also depends on amplification of the signal during the chemical reaction, and it is the enzyme that amplifies the signal. Over the years several types of chromogenic substrates that produce a visible color change have been developed. The newer EIA techniques use electrochemiluminescence, fluorogenic and quantitative polymerase chain reactions to obtain quantifiable signals. These newer substrates have many advantages including higher sensitivity and multiplexing. There are several types of substrates that are used for chemiluminescence, but the most popular type is luminol or its derivatives.


What are the benefits of chemiluminescent immunoassays?


Compared to other immunoassays, chemiluminescence is much more sensitive and can detect a very small amount of a foreign protein, antigen or a biological molecule in a sample. Further, there is a good linear correlation between the luminous intensity and the concentration of the measured antigen. The biochemical reaction can further be amplified by the addition of an enhancer, which is usually an enzyme that permits the reaction to occur for a much longer time without causing any decrease in the output of light. The amplified biochemical reaction generates a strong light emission for a prolonged time period, as well as the enzyme/substrate only needs to be added a few minutes prior to detection.


Practical applications of chemiluminescence


Pharmaceutical Industry (analysis and quality control). The technique of chemiluminescence is widely used in the pharmaceutical industry to screen for contamination of biological compounds and check for impurities in drugs. In addition, the technique is also used to measure levels of hormones. Finally chemiluminescence is also used to detect many drugs in body fluids.


Clinical Science: In the clinical laboratory, chemiluminometric assays based either coupled or direct biochemical reactions utilizing NADPH, ATP, or H2O are common. The technique is also used to measure a variety of hormones, proteins and peptides


In the Alumina Industry, transparent aluminium oxide is used in some lamps that contain sodium vapor. The aluminum oxide is used to as a coating suspension in some compact fluorescent lamps.


In forensic medicine, chemiluminescence is often used to check for presence of blood at a crime scene. In many crimes, blood may not be visible because the criminal may have washed the area, but scientists can use the luminol reaction to locate traces of blood that would not be detectable with the naked eye. The light emitted via the luminol reaction occurs when the blood catalyzes the oxidation of luminol when hydrogen peroxide is applied. The other advantage of luminol is that it can be used to highlight blood on almost any surface and even many days or weeks later.


High-Performance Liquid Chromatography (HPLC). Over the past 3 decades, progress has been made in the development of HPLC detection systems based on chemiluminescence. Today the technique of HPLC using chemiluminescence cna help detect molecules in the femtogram to picogram range with great accuracy.


Chemiluminescence in cancer: Today one of the major causes of death is cancer. There are many types of cancer and one of the major problems with most cancers is a delayed diagnosis. The available treatments do not work well when the cancer is advanced and thus most of the research is geared towards early identification of the cancer, where the chances of cure are very high. The current state of art in chemiluminescence is now being applied in the field of oncology to help detect cancer. In addition, the same technique is now being used to check for presence of recurrence after treatment. Chemiluminescence can identify many types of cancer molecules and other markers much early, thus offering patients a chance for cure. With early treatment, the life expectancy is also increased. The other benefit of chemiluminescence in oncology is that it can be used to identify cancer cells and other markers not only in the tissues but a variety of body fluids. The biggest advance of chemiluminescence in oncology is that it is now offering physicians to direct photodynamic therapy as a light delivery system. For example, the body is screened for areas of chemiluminescence and therapy can be delivered locally, which intensifies the treatment and also avoid systemic toxicity of the cancer drugs.


Wider applications in medicine: While in the past chemiluminescence has traditionally been used to assess the oxidative mechanisms of white blood cells, the technique has seen much wider application in the past two decades. Today chemiluminescence is used for protein blotting, immunoassay, pharmacological and toxicological testing. Further the technique is also used to detect trace amounts of gas like nitric oxide and sulfur. Sulfur compounds, levels of arsine and ozone are now being detected in the environment with great accuracy.