What is the Difference Between Colorimetric and Fluorometric Assay

The main difference between colorimetric and fluorometric assay is that colorimetric assay is less sensitive, whereas fluorometric assay is more sensitive.

Colorimetric and fluorometric assays use a colored compound or fluorescence compound to determine the concentration of a particular chemical compound. An instrument can be used to detect the response.

Key Areas Covered

1. What is Colorimetric Assay
     – Definition, Features, Importance
2. What is Fluorometric Assay
     – Definition, Features, Importance
3. Similarities Between Colorimetric and Fluorometric Assay
     – Outline of Common Features
4. Difference Between Colorimetric and Fluorometric Assay
     – Comparison of Key Differences

Key Terms

Colorimetric Assay, Fluorometric Assay

What is Colorimetric Assay

Colorimetric assays are techniques that determine the concentration of colored compounds in a sample. A colorimeter is an instrument involved in the measurement of the color development in the sample. It measures the absorbance of a specific wavelength of light. Its sample can contain different biomolecules such as enzymes, hormones, antibodies, and other analytes that can develop color. Therefore, in colorimetric assays, the development of color measures the presence and concentration of a particular analyte.

Figure 1: ELISA

Furthermore, examples of colorimetric assays include:

• para-Nitrophenylphosphate – Alkaline phosphatase converts para-Nitrophenylphosphate into the yellow color product.
• Coomassie Blue – An aromatic dye that binds positively charged amino acid residues within the protein structure and aromatic proteins. It quantitatively measures the protein concentration in a sample.
• Bicinchoninic acid assay – Another assay to measure protein concentration.
• Biuret assay – Determines the presence of proteins by turning them into purple color.
• Enzyme-linked immunoassays (ELISA) – Uses to detect antigens by enzyme-linked antibodies.

What are Fluorometric Assay

Fluorometric assays are a type of assay that measures any parameter using fluorescent emission. Fluorometry is important in measuring concentrations and the presence of an analyte in samples by emitting fluorescence. Fluorescence can detect cell organelles such as cell membranes, cell walls, and nuclei and biomolecules such as nucleic acids and proteins. Moreover, fluorescence is a substance that absorbs light emitting light in higher wavelengths. Monarch caterpillars, jellyfish, corals, some sharks, and scorpions are some organisms that naturally emit fluorescence.

Figure 2: Fluorescence Assays

Moreover, fluorescence is important in the qualitative and quantitative measurement of a single type of biomolecules, cell organelles, or cells. DNA-protein or Protein-protein interactions are vital in the fluorometric assays. In addition, fluorescent probes, as well as fluorescence dyes, can be used in the fluorometric assays.

Similarities Between Colorimetric and Fluorometric Assay

• Colorimetric and fluorometric assays are two types of assays important for the determination of concentrations of chemical compounds in samples.
• An instrument can detect the amount of color or fluorescence generated in a sample.

Difference Between Colorimetric and Fluorometric Assay

Definition

Colorimetric assay refers to the use of reagents to measure the color change in the presence of an analyte, while fluorometric assay refers to the technique of measuring any parameter using fluorescent emission.

Significance

A colorimetric assay measures the development of color, while a fluorometric assay measures the development of fluorescence.

Importance

Colorimetric assays detect the presence of enzymes, antibodies, hormones, and other specific compounds in a sample, while fluorometric assays detect the presence of biological molecules such as proteins.

Sensitivity

Colorimetric assays are less sensitive, while fluorometric assays are more sensitive.

Conclusion

In brief, colorimetric and fluorometric assays are two types of assays important for the detection of the presence and concentration of biomolecules. Colorimetric assays develop color in the presence of certain biomolecules such as enzymes, hormones, antibodies, and other specific compounds. However, colorimetric assays are less sensitive. In comparison, fluorometric assays develop fluorescence in the presence of certain biomolecules, such as proteins. They are also more sensitive. Therefore, the main difference between colorimetric and fluorometric assays is their sensitivity.

References:                     

1. “Colorimetry.” An overview | ScienceDirect Topics.
2. “All about fluorometric detection assays.” GoldBio. 

Image Courtesy:

1. “ELISA TMB” By Ajpolino – Own work (CC0) via Commons Wikimedia
2. “Multicolor fluorescence image of living HeLa cells” By 8x57is – Own Work (CC-BY SA 4.0) via Commons Wikimedia