Beadlite™ Rapid Colorimetric Amino Quantitation Kit for Nanoparticles

Surface functionalized micro- and nano-particles have been widely used in the field of nanotechnologies and biological sciences such as bio-separation and purification, assay development, and drug delivery. Among the common functional groups, amine group is one of the most popular groups for modification, and is often the first active group to be introduced. Various biomolecules, either probe molecules or target molecules, can thus be immobilized on the surface of nanoparticles through the reaction with these amine groups. The density of amine groups plays a key role in determining the properties of nanomaterials and controlling their interactions with biomolecules. Therefore, quantitation of amine density on the surface of particles is critical to their applications. Beadlite™ Rapid Colorimetric Amino Quantitation Kit offers a rapid and sensitive absorption-based assay for measuring amine density with high specificity. Upon the selective reaction with the amine group, the Bucculite™ MTA is first immobilized on the surface of particles, and then the modified particles are added to Bucculite™ MTA-Dye 650 solution resulting in a decrease of absorbance in supernatant. The signal can be measured by UV-Vis spectrometer at ~650 nm and the change is proportional to the amount of amine groups on the surface of the nanoparticles.

Example protocol

AT A GLANCE

Protocol summary

Wash beads
Mix beads and Component A
Rotate 1 hour at room temperature
Wash beads
Prepare Component B solution and measure absorbance at 650 nm as “Original absorbance”(A_O)
Mix beads and Component B solution
Rotate 1.5-2 hours at room temperature
Measure absorbance of supernatant at 650 nm as “Remaining absorbance”(A_R)
Calculate amine density on beads

Important Note

Thaw all the kit components at room temperature before starting the experiment.

SAMPLE EXPERIMENTAL PROTOCOL

Take 1 mg beads that need to be tested. Wash beads with 0.5-1 mL Assay Buffer (Component D) for 3 times and remove all supernatant.
Disperse beads in 100 µL Reaction Buffer (Component C). Add this suspension to one vial of Component A and mix well.
Rotate the vial (from step 2) at room temperature for 1 hour.
Wash beads with 0.5-1 mL Assay Buffer (Component D) for 3 times and remove supernatant.
Add 100 µL Assay Buffer (Component D) to one vial of Component B to make Component B stock solution.
Take 2 µL Component B stock solution (from step 5) to 198 µL of Assay Buffer (Component D) to have 1 to 100 x dilution. Measure absorbance at 650 nm as “Original absorbance” (A_O).
Disperse beads in the rest 98 µL of Component B stock solution.
Rotate it (from step 7) at room temperature for 1.5- 2 hours.
Separate beads and supernatant.
Take 2 µL of supernatant (from step 9) to 198 µL Assay Buffer (Component D) to have 1 to 100 x dilution. Measure absorbance at 650 nm as “Remaining absorbance”(A_R).

Calculate amine density on beads:

Amine density (nmol/mg) = (Original absorbance-Remaining absorbance) * 39.2 nmol/mg

Page updated on November 21, 2024

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