Statistical and generalized two-dimensional correlation spectroscopy of multiple ionization States. Fluorescence of neurotransmitter serotonin

Fluorescence spectra of neurotransmitter serotonin are analyzed with generalized and statistical two-dimensional correlation spectroscopy. A comparison is provided for these two emerging data analysis techniques. Both methods reveal correlations between spectral variables and demonstrate enhanced sensitivity in detecting the dynamic spectral changes over conventional one-dimensional spectroscopy. Both statistical and generalized 2D correlation analysis emphasize simultaneous spectral changes in response to external perturbations. Generalized 2D correlation spectroscopy further reveals the difference in rates of these dynamic changes. Using 2D correlation analysis, a third ionization species of serotonin is identified using pH and excitation wavelength perturbation. This species is a doubly deprotonated serotonin with very low fluorescence quantum yield, confirmed by using a laser excitation at longer wavelength and at higher pH. Taking advantage of the spectral differences between excitation of serotonin and tryptophan, as low as 3.8 nM serotonin can be detected in the presence of 20 microM tryptophan, with long-wavelength excitation. This represents the sensitive detection of serotonin in 5000-fold excess of tryptophan.