Colorimetric quantification of amino groups

 Colorimetric quantification of amino groups

Preparation of standard solutions
Stock solutions of BSA, poly(Lys), poly(Arg), poly(Pro), poly(His),
DGL (generations 1, 2, 3, 4, 5), PEI and PAMAM dendrimers were
prepared at 100 μg mL −1 in water (18 M ) purified from a MilliQ UV
Plus Millipore system. Standard solutions were all freshly prepared
from the above stock solutions avoiding successive dilutions.
Water mixtures of the poly(amino acid)s or others macromolecules
were at a final concentration of 0.2 to 40 μg mL −1 when processing
the microplate modified Bradford assay as explained in the
following section.

Microplate procedure
Assays were carried out in 96 native microplate format wells
as described by the supplier. The dye reagent was added to
the stock macromolecule solution containing protein, linear or
dendritic structures so that the dye concentration (D T ) was
maintained constant in 300 μL final volume per well with 1 : 1
CBB solvent : water mixture volume. After mixing the solution
and waiting for 5 min in order to allow maximum dye binding,
the absorbance was monitored at 620 nm. All experiments were
repeated at least three times per sample concentration. When
adding a macromolecule mixture to the dye solution, the observed
change in absorbance ( A) is then given by the following equation
based on the Lambert–Beer law:

deltaA = (alpha Bound − alpha Free )/D Bound

where alpha Bound and alpha Free are species molar absorptivities for bound
and free dye under the microplate assay conditions with pH being
about 1, D Bound is the concentration of bound dye and l is the
optical length path.

Determination of molar absorptivities
To optimize binding of selected macromolecule sites with dye,
we carried out experiments at various dye/macromolecule ratios.
We prepared a modified Bradford reagent following a procedure
reported by Peterson. 10 Briefly, CBB (20.5 mg) was dissolved in
95% ethanol (10 mL) followed by the addition of 85% phosphoric
acid (20 mL). This mixture was completed with ultrapure water to
120 mL and sonicated. Solutions containing dye concentrations
between 2 × 10 −5 and 2 × 10 −4 mol L −1 were prepared from
the modified reagent. Free anion dye molar absorptivity (a Free )
was estimated from a Beer’s law plot considering an optical path
length l of 1.05 cm (well volume solution high). The a Free value was
14 199 L mol −1 cm −1 . The obtained data were processed using
a simplified Atherton mathematical model considering that only
CBB blue species are taken into account during binding so that
a CBB blue species = a Bound . 5 A double reciprocal plot representation
was drawn to provide a value of the absorptivity factor ( a)
and molar absorptivities of macromolecule-bound dye (a Bound ).
Molar absorptivities were calculated under excess macromolecule
conditions