News & Blogs 2024-08-13
NHS and Sulfo-NHS and Their Impact on Reagent Performance
Brief Introduction of Magnetic Microsphere Activators
Magnetic microsphere activators are chemical reagents used to introduce active groups on the surface of magnetic beads, enhancing the binding efficiency between the beads and target molecules (such as antibodies, antigens, proteins, etc.). The main functions of activators include:
● Enhancing Binding Efficiency: By introducing active groups, the binding efficiency and sensitivity between magnetic beads and target molecules are improved.
● Reducing Non-Specific Binding: Reducing non-specific reactions, thereby improving the accuracy of detection results.
● Multi-Functionalization: Allowing the surface of the magnetic beads to connect with various types of molecules, achieving multiple labeling and detection.
Differences Between NHS and Sulfo-NHS
NHS (N-Hydroxysuccinimide) and Sulfo-NHS (N-Hydroxysulfosuccinimide) are two common activators used to convert carboxyl (COOH) groups into more reactive ester groups for cross-linking reactions with amino (NH2) groups. Their main differences are as follows:
Item | Molecular Weight | CAS # | Structural formula |
NHS (N-HYDROXYSUCCINIMIDE) | 115.09 | 6066-82-6 |  |
Sulfo-NHS (N-HYDROXYSULFOSUCCINIMIDE) | 217.13 | 106627-54-7 |  |
source:
https://www.chemicalbook.com/CAS_6066-82-6.htm
https://www.chemicalbook.com/CAS_106627-54-7.htm
The Special Functions of NHS (N-Hydroxysuccinimide)
NHS is well-known in the in vitro diagnostic field, forming a "golden duo" with EDC in the microsphere activation process, highly praised by many researchers. Its main features include:
● NHS transforms carboxyl groups into NHS esters that can react with amino groups, effectively acting as a catalyst and significantly improving coupling efficiency.
● In the presence of EDC, NHS enhances the efficiency of the reaction with amino groups, thereby increasing coupling efficiency.
● NHS is insoluble in water, usually used dissolved in organic solvents (such as DMF or DMSO), and possesses membrane permeability.
The Unique Traits of Sulfo-NHS (N-Hydroxysulfosuccinimide)
Sulfo-NHS, derived from NHS, functions similarly with additional benefits:
● Like NHS, Sulfo-NHS can also convert carboxyl groups into NHS esters.
● In the presence of EDC, it similarly enhances the reaction efficiency with amino groups.
● Sulfo-NHS, containing a sulfonate group, is more water-soluble and less likely to cause aggregation of conjugates due to its negative charge.
● Sulfo-NHS dissolves poorly in organic solvents and lacks membrane permeability, but it is highly water-soluble and can be used directly in water or buffer solutions.
Differences in Microsphere Activation Using NHS and Sulfo-NHS
Water Solubility Differences After Microsphere Carboxyl Group Modification
Despite both being activators, NHS and Sulfo-NHS show differences in practical microsphere activation processes. For example, carboxyl microspheres, which inherently have good hydrophilicity due to numerous carboxyl functional groups on their surface, show reduced water solubility after NHS modification, whereas Sulfo-NHS-modified microspheres maintain or even improve their original water solubility due to the hydrophilicity of the sulfonate group.
Impact on Reagent Sensitivity and Linearity
The structural and functional differences between NHS and Sulfo-NHS lead to different coupling results. In an experiment with an inflammation-related project on a fluorescence immunochromatography platform, microspheres were coupled with NHS and Sulfo-NHS under the same conditions using EDC. The results showed varying linearity and sensitivity:
● NHS provided higher sensitivity at low values, with linearity reaching a plateau earlier.
● Sulfo-NHS offered a broader linear range but lower sensitivity at low values.
These phenomena may vary depending on the project, as activators fundamentally affect the amount of protein coupled to the microsphere surface, leading to differences in linear performance.
How to Choose?
● If NHS repeatedly adjusted does not achieve the desired high-value linearity, consider trying Sulfo-NHS.
● If Sulfo-NHS fails to meet low-value sensitivity requirements, consider trying NHS.
● If neither works for low-value sensitivity or high-value linearity, consider using larger microspheres or replacing the labeled antibody, etc.