Serotonin-Derived Fluorophore: A Novel Fluorescent Biomaterial for Copper Detection in Urine

Abstract

We took advantage of the fluorescent features of a serotonin-derived fluorophore to develop a simple and low-cost assay for copper in urine. The quenching-based fluorescence assay linearly responds within the concentration range of clinical interest in buffer and in artificial urine, showing very good reproducibility (CV_av% = 4% and 3%) and low detection limits (16 ± 1 μg L^-1 and 23 ± 1 μg L^-1). The Cu²⁺ content was also estimated in human urine samples, showing excellent analytical performances (CV_av% = 1%), with a limit of detection of 59 ± 3 μg L^-1 and a limit of quantification of 97 ± 11 μg L^-1, which are below the reference value for a pathological Cu²⁺ concentration. The assay was successfully validated through mass spectrometry measurements. To the best of our knowledge, this is the first example of copper ion detection exploiting the fluorescence quenching of a biopolymer, offering a potential diagnostic tool for copper-dependent diseases.

Keywords: copper detection; copper poisoning; copper-dependent diseases; fluorescence quenching; serotonin; serotonin-derived fluorophore; urine analysis.

Scheme 1

(a) Putative SEDF structure obtained by SE monomer polymerization upon linkage through the benzene ring after 2 h at 60 °C in 10 mM TRIS buffer pH 9; (b) Cu²⁺ detection in a 96−well microplate by reading the SEDF fluorescence quenching upon a [Cu²⁺] increase in urine.

Figure 1

(a) UV-Vis spectra of the SE monomer (red line, right cuvette) and synthetic SEDF (black line, left cuvette) in 10 mM TRIS pH 9.00; (b) fluorescence excitation (red, λ_em = 450 nm) and emission (black, λ_ex = 350 nm) spectra of serotonin-derivative fluorophore (SEDF, thick line) and serotonin (SE, dashed line).

Figure 2

MALDI-TOF/TOF mass spectrum of the SE-derivative fluorophore (SEDF) obtained by heating 2 g L⁻¹ serotonin at 60 °C for 2 h in 10 mM TRIS at pH 9.00. The m/z values compatible with SE monomer (a), dimer (b), trimer (c), and tetramer (d) molecules are reported.

Figure 3

Fluorescence emission (λ_ex = 350 nm, λ_em = 450 nm) of serotonin-derivative fluorophore (SEDF) obtained by heating 2.0 g L⁻¹ SE monomer. The influence of pH 2–12 (a,b), temperature 40 °C–90 °C (c,d), and reaction time 0.5–5 h (e,f) on the fluorescence emission spectra (left) and intensity at 450 nm (right). The error bars represent the standard deviation (n = 4).

Figure 4

Influence of the serotonin monomer concentrations (0, 0.001, 0.005, 0.010, 0.050, 0.100, 0.200, 0.300, 0.400, 0.500, 1.000, and 2.000 g L⁻¹) on the emission signal of the serotonin−derivative fluorophore synthesized after 2 h at 60 °C in alkaline conditions (pH 9.00). The fluorescence emission spectra upon excitation at 350 nm (a) and corresponding fluorescence emission intensity at 450 nm (b). The error bars represent the standard deviation (n = 4).

Figure 5

Fluorescence of the SEDF in 10 mM TRIS at pH 9.00 after the addition of 0.5 mM alkaline and alkaline earth metals (a), transition metals (b,c), and organic compounds occurring in urine (d). The error bars represent the standard deviation (n = 4).

Figure 6

Fluorescence spectra and the relative calibration plots of the SEDF after copper ion addition in buffer (a,c) and in artificial urine samples (b,d). The fluorescence was recorded at 450 nm (λ_ex = 350 nm) and reported as a F₀/F versus Cu²⁺ concentration (0, 0.010, 0.050, 0.100, 0.165, 0.250, 0.350, and 0.450 mM), where F₀ and F represent the fluorescence intensity of the SEDF before and after the copper addition, respectively. The error bars represent the standard deviation (n = 4). The analytical parameters are reported in Table S1.

Figure 7

Calibration of Cu²⁺ in human urine (sample 3, red dots and fitting) and its quantification in the four urine samples, indicated by the different colors (1—green, 2—violet, 3—black, and 4—blue). The fluorescent signal is reported as F₀/F at λ_em = 450 nm (λ_ex = 350 nm) versus the Cu²⁺ concentration, where F₀ and F represent the fluorescence intensity of the SEDF before and after the copper addition, respectively. The error bars represent the standard deviation (n = 4). The analytical parameters of the calibration are reported in Table S2 and were obtained using the piecewise linear fitting implemented in the OriginPro software, version 2022. OriginLab Corporation, Northampton, MA, USA.