What is Biotinylation? New Breakthrough Applications in 2025

Publish Time: 2025-05-19

Biotinylation: An Overview

Biotinylation, as defined by the American Chemical Society (ACS) is the process of covalently attaching biotin, a small vitamin B7 derivative (MW = 244.31 g/mol), to a protein, nucleic acid, or other molecule. This technique is popular due to its rapidity, specificity, and minimal interference with the natural function of the modified molecule.

Figure 1: Chemical structure of biotin

Biotinylation allows for the precise labeling of biomolecules, making it an invaluable tool in various scientific disciplines. See this Wikipedia article for a comprehensive introduction.

How Does Biotinylation Work?

The magic of biotinylation lies in the extremely high affinity between biotin and proteins like streptavidin and avidin. According to Thermo Fisher Scientific biotin binds to streptavidin and avidin with a dissociation constant (Kd) of approximately 10⁻¹⁴ mol/L. This strong interaction allows biotinylated molecules to be captured or detected under extreme conditions.

Figure 2: Structural diagram of the reaction between biotin-NHS and lysine

A common method uses biotinylation reagents such as N-hydroxysuccinimide (NHS) esters to target primary amines. Once the molecule is biotinylated, it can be easily captured or detected using streptavidin-coated surfaces or streptavidin-conjugated probes.

What Are the Uses of Biotinylation?

In Research

Biotinylation is essential in molecular biology. Techniques such as affinity chromatography use biotinylated proteins to isolate specific targets. As explained in the Journal of Chromatography A this method improves purification precision.

Biotinylation is also used in protein–protein interaction studies and pull-down assays, as highlighted in Nature Methods.

In Diagnostics

Biotin-streptavidin technology enhances sensitivity in immunoassays, such as ELISAs. Guidelines from the Clinical and Laboratory Standards Institute (CLSI) confirm the effectiveness of biotinylated antibodies in detecting low-level biomarkers.

In Therapeutics

Biotinylated nanoparticles have shown promise in targeted drug delivery. A study funded by the National Institutes of Health (NIH) used biotinylated liposomes for targeting cancer cells with encouraging preclinical outcomes.

Biotinylation Reagents and Solubility