Given only two major absorbing species in the complex system,
the spectra obtained at the beginning and end of the evolution
process are selected as initial estimates of the later ALS iterative
optimizations. Concomitantly, some constraints, such as nonnegativity for both concentration evolutions and spectra,local
rank information for concentration evolutions and normalization
for spectra, are introduced in the optimization process to reduce
rotational ambiguity. The optimum relative normalized concentration evolutions of pure species estimated with MCR-ALS
method are presented in Fig. 4A.The quality parameters for the
resolution give a lack of fit of 0.16% and a variance explained of
99.99%, suggesting that perfect resolution results are gained. The
distribution diagram clearly shows that the relative concentration
of AuNPs decreases significantly ,that of HSA–AuNPs conjugate
increases sharply and their concentration sreachsaturation at
11:1 molar binding stoichiometry between HSA and AuNPs