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

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 (CVav% = 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 (CVav% = 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.


Introduction
Copper ion is essential to living systems, regulating many physiological functions, acting as a cofactor of numerous enzymes (e.g., dopamine β-hydroxylase, tyrosinase, and cytochrome c oxidase [1]), contributing to cellular and tissue growth, and working as an antioxidant [2]. The World Health Organization (WHO) recommends an intake of 30 µg per kilogram of body weight per day [3]. However, an excess of this heavy metal leads to protein structure modification, interfering with the exchange of zinc in metalloproteins and compromising most cellular functions [4]. Recently, it was demonstrated that an accumulation of Cu 2+ affects the activity of dopaminergic neurotransmitters, potentially leading to neurodegenerative disorders and mental issues, including anxiety, depression, language, and cognitive impairment [5][6][7]. In this context, a few fluorescent methods were developed for Cu 2+ quantitative analysis in urine [8][9][10][11][12][13][14]. In this study, for the first time, the fluorescent properties of a novel fluorophore derived from serotonin (SE) self-oxidation and polymerization were exploited for sensitive Cu 2+ detection in human urine without matrix pretreatment. SE is a neurotransmitter involved in multiple important physiological processes, such as memory, learning, anxiety, depression, cognition, vomiting, and vasoconstriction [15]. Very few studies describe the formation of SE oligomers [16][17][18][19], including polyserotonin (PSE) obtained using horseradish peroxidase (HRP) [19], and serotonin-based nanoparticles (PSE-NPs) [16,17] as multifunctional material for free radical scavenging, bioelectrical, and biomedical applications. In this paper, the highly emitting SE-derivative fluorophore (SEDF) was obtained by heating the SE monomer at an alkaline pH (Scheme 1a). SEDF was characterized by means of UV-Vis spectroscopy and mass spectrometry. The fluorescent properties of SEDF were outstanding when compared with the SE-derivative fluorophore (SEDF) was obtained by heating the SE monomer at an alkaline pH (Scheme 1a). SEDF was characterized by means of UV-Vis spectroscopy and mass spectrometry. The fluorescent properties of SEDF were outstanding when compared with the fluorescent properties of polymers obtained using other endogenous neurotransmitters, i.e., polydopamine (PDA) and polynorepinephrine (PNE) [20,21]. The analytical performances of the fluorescence detection strategy were firstly evaluated by determining the Cu 2+ in buffer and in artificial urine. Then, based on these results, we proceeded to determine the Cu 2+ concentration in human urine samples (Scheme 1b) observing the reference values sufficient to detect the early stage of diseases associated with Cu 2+ accumulation, such as chronic liver disease, acute hepatitis, and, primarily, Wilson's disease (WD), a rare inherited disorder that can lead to excess storage of copper in the liver, brain, and other organs [22,23]. Patients who suffer from WD have high urinary copper values between ca. 200 and 400 μg per day [24,25]. Values of Cu 2+ higher than 100 μg per day are strongly indicative of this disease [23]. Common diagnostic assays for WD are based on the ceruloplasmin (Cp) concentration and on hepatic and urinary Cu 2+ detection [23,26]. However, the Cp test is inaccurate for the estimation of free Cu 2+ , whereas the evaluation of hepatic copper requires an invasive procedure and, often, the heterogeneous Cu 2+ distribution within the liver leads to false negative results. Differently, the experimental procedure here developed for copper estimation would be noninvasive, fast, very simple, green, and low cost if compared to the reference instrumental approach for copper quantification in urine samples, such as inductively coupled plasma mass spectrometry (ICP-MS) [27], here used to validate the fluorescence method. This work aims to propose a new diagnostic tool for copper-dependent diseases, representing, to the best of our knowledge, the first example of copper ions detection exploiting the fluorescence quenching of a biopolymer. 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) Cu 2+ detection in a 96well microplate by reading the SEDF fluorescence quenching upon a [Cu 2+ ] increase in urine.

Commented [M6]
center dot 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) Cu 2+ detection in a 96−well microplate by reading the SEDF fluorescence quenching upon a [Cu 2+ ] increase in urine.

Instrumentation
The temperature-controlled synthesis reaction of SEDF was performed using a Thermomixer comfort (Eppendorf, VWR International, Milan, Italy). The fluorescence experiments were performed with fluorimeter FP-6500 (Jasco, Easton, PA, USA) using an excitation and emission wavelength of λ ex = 350 nm and λ em = 450 nm, respectively (emission bandwidth: 10 nm; excitation bandwidth: 5 nm; data pitch: 1 nm; scanning speed: 100 nm min −1 ; sensitivity: low) and microplate readers Fluoroskan Ascent (Thermo Fisher Scientific, Milan, Italy) by selecting as filter pairs λ ex = 390 nm and λ em = 460 nm, corresponding to the excitation and emission wavelengths, respectively. The absorbance measurements were obtained using a SPECTROstar Nano UV-Visible Spectrophotometer (Ortenberg, Germany) in 1.0 cm quartz cells at 20 • C. The mass spectrometry measurements, acquired in the positive-ion mode, were performed with a MALDI-TOF/TOF Ultraflex III (Bruker Daltonics, Milan, Italy) (matrix-assisted laser desorption/ionization time-offlight mass spectrometry) by mixing the samples in a 1:1 ratio with 20 ng µL −1 DHB (2,5-dihydroxy benzoic acid) as matrix dissolved in 70% of acetonitrile, 25% trifluoroacetic acid, and 5% ethanol. The mass spectra were acquired in the positive-ion mode.

Synthesis of Serotonin-Derived Fluorophore
A total of 2 g L −1 of serotonin solution was dissolved in a 10 mM TRIS buffer pH 9.00 and heated at 60 • C for 2 h. Subsequently, the sample was left for 10 min at room temperature, then centrifuged 2 times for 10 min at 10,000 rpm. Finally, the supernatant was collected and stored at 4 • C after the addition of 5 mM HCl.

Quantum Yield Calculation
The fluorescence quantum yield (Q) of the SEDF was estimated using quinine sulfate dissolved in 0.1 M H 2 SO 4 as the fluorescence reference standard of the known quantum yield (Q R ), as reported by Lakowicz [28]. The quantum yield of the SEDF was calculated using the following equation: where I is the integrated fluorescence intensity, OD is the optical density at the excitation wavelength, and n is the refractive index of the solvent. The subscript R indicates the same parameters for the reference fluorophore, in this case quinine sulfate. The absorbances at the wavelength of excitation (λ ex = 350 nm) were kept at A = 0.05 to avoid the inner filter effect [28], thus ensuring a linear dependence of the fluorescence signals to the sample concentration.

Copper Determination via the Fluorescence Quenching-Based Method
Stock Cu 2+ solutions were prepared in 10 mM Tris pH 9.00 and in artificial urine. The different volumes of copper solution were mixed with solutions of previously synthesized SEDF (see above), immediately recording the fluorescence signal. The decrease in the fluorescence in dependence on the copper concentration was expressed as F 0 /F, where F 0 is the fluorescence of the SEDF without any metal addition, and F is the fluorescence recorded after the copper addition in buffer, artificial urine, and real urine samples. All measurements were performed at 25 • C in quadruplicates, at least. The limit of detection (LOD) and limit of quantification (LOQ) were calculated by applying the following equations: LOD = 3 × SD blank /slope and LOQ = 10 × SD blank /slope, where the standard deviation of the blanks (SD blank ) was divided by the slope of the relative calibration plots.

ICP-MS Measurements
The method here developed was validated using an Agilent Technologies 7900 ICP-MS (Santa Clara, CA, USA) equipped with an ASX-500 Series autosampler and a peristaltic pump for the sample injection. The analyses were performed with the following acquisitions parameters: carrier gas flow rate of 0.80 L min −1 (Ar), aerosol dilution flow rate of 0.50 L min −1 (Ar), plasma gas flow rate of 15 L min −1 (Ar), collision gas flow rate of 4.3 × 10 −3 L min −1 (He), RF power of 1550 W, stabilization time of 20 s, peak pattern of 3 point, 3 replicates, 100 sweeps per replicate, and a peristaltic pump speed of 0.1 rps. The system control and data acquisition and processing were carried out using the ICP-MS MassHunter Workstation ® software, version 5.1. For the pre-analytical procedure, the Agilent environmental calibration standard mix, including copper ( 63 Cu) at a concentration of 10,000 µg L −1 , was diluted in deionized water to prepare an initial calibration curve ranging from 0 to 2500 µg L −1 . The calibration points at 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 250 µg L −1 were obtained by dilution with water:1-butanol = 98.5:1.5 (v/v) added with Triton X100, 10 µL L −1 , and Tetramethylammonium hydroxide solution (TMAH 25% in H 2 O), 100 µL L −1 . Each calibration point was added with the appropriate amount of germanium ( 72 Ge) as the internal standard. Two quality control points were added to attest the accuracy of the analysis. The urine samples, previously centrifuged at 1230 rpm for 15 min to remove the sediment, were analyzed after a dilution of 1:100 with water: 1-butanol solution with the appropriate amount of the internal standard.

Synthesis and Characterization of Serotonin-Derived Fluorophore (SEDF)
Recently, Jeon et al. reported the synthesis of insoluble polyserotonin nanoparticles from the self-oxidation of the SE monomer heated for 2 h at 60 • C in TRIS buffer at pH 9.00 [16]. Repeating the same procedure, we collected the supernatant solution instead of the precipitated nanoparticles, thus obtaining the serotonin-derived fluorophore (SEDF). The SEDF solution was brown in color with a λ max = 470 nm, indicative of a larger conjugated system with respect to the serotonin monomer showing the spectrum typical of a colorless indole derivative with maximum absorbance values at 280 nm and 300 nm ( Figure 1a). Analogously, the fluorescence spectra of the SEDF showed a large emission band of approximately 450 nm, whereas in the same conditions the SE monomer was not fluorescent (Figure 1b).
Previous works refer to an SE dimer [29,30], while others describe an oligomer obtained through the electrochemical oxidation of serotonin [31][32][33]. To better understand the nature of the serotonin derivative obtained here, we performed mass spectrometry experiments by MALDI-TOF/TOF instrumentation. The peaks highlighted in Figure 2 (m/z 176.689, 350.978, 502.098, and 672.180) are compatible with monomeric, dimeric, trimeric, and tetrameric structures, possibly obtained upon the linkage through the benzene ring (Scheme 1) [19]. However, the determination of the SEDF structure is out of the scope of this study and requires a much deeper analysis.  Previous works refer to an SE dimer [29,30], while others describe an olig tained through the electrochemical oxidation of serotonin [31][32][33]. To better un the nature of the serotonin derivative obtained here, we performed mass spec experiments by MALDI-TOF/TOF instrumentation. The peaks highlighted in (m/z 176.689, 350.978, 502.098, and 672.180) are compatible with monomeric, dim meric, and tetrameric structures, possibly obtained upon the linkage through the ring (Scheme 1) [19]. However, the determination of the SEDF structure is out of of this study and requires a much deeper analysis. The influence of the synthetic parameters, such as pH, reaction time, and monomer concentration, on the fluorescent properties of the SEDF were tested ( and, notably, the best fluorescence performances of the supernatant coincide wit conditions for the size-controlled synthesis of polyserotonin nanoparticles [16].
In detail, the influence of the pH on the fluorescence of the SEDF was eval tween 2.00 and 12.00. The solutions of the SEDF when excited at 350 nm showed spectra with a λmax of approximately 400 nm at pH 2.00-4.00, 425 nm at pH 6.00-450-475 nm at pH 8.00-12.00 (Figure 3a,b). The intensity of the fluorescence emi  Previous works refer to an SE dimer [29,30], while others describe an oligome tained through the electrochemical oxidation of serotonin [31][32][33]. To better under the nature of the serotonin derivative obtained here, we performed mass spectrom experiments by MALDI-TOF/TOF instrumentation. The peaks highlighted in Fig  (m/z 176.689, 350.978, 502.098, and 672.180) are compatible with monomeric, dimer meric, and tetrameric structures, possibly obtained upon the linkage through the ben ring (Scheme 1) [19]. However, the determination of the SEDF structure is out of the of this study and requires a much deeper analysis. The influence of the synthetic parameters, such as pH, reaction time, and sta monomer concentration, on the fluorescent properties of the SEDF were tested (Fig  and, notably, the best fluorescence performances of the supernatant coincide with th conditions for the size-controlled synthesis of polyserotonin nanoparticles [16]. In detail, the influence of the pH on the fluorescence of the SEDF was evaluate tween 2.00 and 12.00. The solutions of the SEDF when excited at 350 nm showed em spectra with a λmax of approximately 400 nm at pH 2.00-4.00, 425 nm at pH 6.00-7.00 450-475 nm at pH 8.00-12.00 (Figure 3a,b). The intensity of the fluorescence emissio The influence of the synthetic parameters, such as pH, reaction time, and starting monomer concentration, on the fluorescent properties of the SEDF were tested (Figure 3) and, notably, the best fluorescence performances of the supernatant coincide with the best conditions for the size-controlled synthesis of polyserotonin nanoparticles [16].
In detail, the influence of the pH on the fluorescence of the SEDF was evaluated between 2.00 and 12.00. The solutions of the SEDF when excited at 350 nm showed emission spectra with a λ max of approximately 400 nm at pH 2.00-4.00, 425 nm at pH 6.00-7.00, and 450-475 nm at pH 8.00-12.00 (Figure 3a,b). The intensity of the fluorescence emission was strongly reduced at a pH below 6.00 or above 11.00. Moreover, highly basic conditions led to heterogeneous mixtures with large scattering phenomena (data not shown). As shown in Figure 3b, the buffer at pH 9.00 resulted in the best fluorescence reproducibility with good fluorescence intensity, and this condition was used for the subsequent studies.
The influence of the temperature on the fluorescence of the SEDF at pH 9.00 for 2 h was evaluated between 40 • C and 90 • C (Figure 3c,d). The fluorescence emission of the SEDF increased with the temperature, and 60 • C resulted in the temperature of choice, representing a good compromise in terms of the signal intensity and reproducibility (Figure 3d). representing a good compromise in terms of the signal intensity and reproducibil ure 3d).
The influence of the reaction time on the fluorescence of the SEDF was ev between 30 min and 5 h at pH 9.00 and 60 °C. The fluorescent spectrum of the SE already visible after 30 min of reaction, reaching the maximum intensity after 1 h 3e,f). However, the best reproducibility was achieved after 2 h of reaction (Figure  The influence of the reaction time on the fluorescence of the SEDF was evaluated between 30 min and 5 h at pH 9.00 and 60 • C. The fluorescent spectrum of the SEDF was already visible after 30 min of reaction, reaching the maximum intensity after 1 h (Figure 3e,f). However, the best reproducibility was achieved after 2 h of reaction (Figure 3f).
Finally, the influence of SE monomer concentration on the fluorescence of SEDF after 2 h at pH 9.00 and 60 • C (Figure 4a,b) was investigated. The largest fluorescence increase was obtained for SE up to 0.5 g L −1 , reaching a plateau at 2 g L −1 SE, which was fixed as the monomer concentration for the following experiments. The synthetic product was stabilized by adding 5 mM of HCl that stopped the oxidation process. The fluorescence intensity in buffer was stable up to 4 h, decreasing to 75% of the starting value after one month ( Figure S1). lized by adding 5 mM of HCl that stopped the oxidation process. The fluorescenc sity in buffer was stable up to 4 h, decreasing to 75% of the starting value after one ( Figure S1).
The fluorescence quantum yield (Q) of the SEDF was estimated in water, as r in the assay protocol, and the obtained value of 0.025 was higher than the Q repo the PSE obtained enzymatically (0.017) [19] and here calculated also for the PDA and PNE (0.008), indicating the good fluorescent properties of such conjugated ser

SEDF Fluorescence Quenching by Metal Ions
The effect of several solutions (0.5 mM) of alkaline, alkaline earth ( Figure 5 transition metals (Figure 5b,c) on the SEDF fluorescence was evaluated, and it wa that only the latter quenched the fluorescence of the SEDF, in particular, Au 3+ , Fe 3+ , and Cu 2+ species (Figure 5c), which is in agreement with literature that report fluorescence quenching of indole derivatives by copper ions and a few other [28,34,35]. The Fe 3+ exhibited a prominent fluorescence quenching, as previously ob for the fluorescent derivatives of dopamine used to quantify iron [36,37]. The fluor quenching was also large for Au 3+ and Cr2O7 2− ions but associated with low data ducibility, which is also due to the formation of gold nanoparticles. Based on these and considering that, to the best of our knowledge, there are no studies on cop detection exploiting the fluorescence quenching of a polymer derived by an endo molecule, such as SEDF, we decided to apply this method to real urine samples the presence of large amounts of copper ions would be indicative of a pathologica tion (see below), whereas the concomitant presence of the other quenching ions, in ular Au 3+ and Cr2O7 2− , is insignificant in urine [38,39], and the urinary level of healthy people (0.2 mg L −1 , 0.003 mM) is approximately two orders of magnitude the concentration of Fe 3+ here tested (0.5 mM), and one order of magnitude below the m amount of Cu 2+ here spiked in urine (0.05 mM, vide infra). However, there is also the p interference of larger urinary levels, as a consequence of other iron-overload d which could be simply minimized by using the proper sequestering buffer, as The fluorescence quantum yield (Q) of the SEDF was estimated in water, as reported in the assay protocol, and the obtained value of 0.025 was higher than the Q reported for the PSE obtained enzymatically (0.017) [19] and here calculated also for the PDA (0.019) and PNE (0.008), indicating the good fluorescent properties of such conjugated serotonin.

SEDF Fluorescence Quenching by Metal Ions
The effect of several solutions (0.5 mM) of alkaline, alkaline earth (Figure 5a), and transition metals (Figure 5b,c) on the SEDF fluorescence was evaluated, and it was found that only the latter quenched the fluorescence of the SEDF, in particular, Au 3+ , Cr 2 O 7 2− , Fe 3+ , and Cu 2+ species (Figure 5c), which is in agreement with literature that reports on the fluorescence quenching of indole derivatives by copper ions and a few other metals [28,34,35]. The Fe 3+ exhibited a prominent fluorescence quenching, as previously observed for the fluorescent derivatives of dopamine used to quantify iron [36,37]. The fluorescence quenching was also large for Au 3+ and Cr 2 O 7 2− ions but associated with low data reproducibility, which is also due to the formation of gold nanoparticles. Based on these results, and considering that, to the best of our knowledge, there are no studies on copper ion detection exploiting the fluorescence quenching of a polymer derived by an endogenous molecule, such as SEDF, we decided to apply this method to real urine samples, where the presence of large amounts of copper ions would be indicative of a pathological condition (see below), whereas the concomitant presence of the other quenching ions, in particular Au 3+ and Cr 2 O 7 2− , is insignificant in urine [38,39], and the urinary level of iron in healthy people (0.2 mg L −1 , 0.003 mM) is approximately two orders of magnitude below the concentration of Fe 3+ here tested (0.5 mM), and one order of magnitude below the minimum amount of Cu 2+ here spiked in urine (0.05 mM, vide infra). However, there is also the possible interference of larger urinary levels, as a consequence of other iron-overload diseases, which could be simply minimized by using the proper sequestering buffer, as already reported [40,41]. Moreover, we also evaluated the interference of the main organic compounds in urine on SEDF fluorescence quenching. As shown in Figure 5d, urea, uric acid, creatinine, and citrate, in the physiological concentration ranges [42], did not lead to a fluorescence quenching. This was also true for glucose, hypoxanthine, glutamic acid, and ascorbic acid at the concentration that determines the pathological state. Accordingly, we proceeded to determine the Cu 2+ concentration in buffer solution, artificial urine, and human urine samples. fluorescence quenching. This was also true for glucose, hypoxanthine, glutami ascorbic acid at the concentration that determines the pathological state. Accor proceeded to determine the Cu 2+ concentration in buffer solution, artificial urin man urine samples.

Copper Quantification via Quenching-Based Bioanalytical Assay
The SEDF fluorescence spectra and dose-response plots in the buffer and i urine (AU) samples are reported in Figure 6. The fluorescence signal was repor where F0 is the fluorescence intensity of the SEDF, and F is the emission signal o after the copper addition. The fluorescence intensity at 450 nm after the Cu 2+ ad plotted versus the Cu 2+ concentration in the range of 0.05-0.5 mM, following a li with remarkable analytical performance (Table S1), both in the buffer and in AU as confirmed by the correlation coefficient R 2 = 0.990 for both and by the lin equations F0/F = 4.68 × [Cu 2+ ] + 0.87 used for the Cu 2+ detection in buffer and F [Cu 2+ ] + 0.93 for AU. The excellent assay reproducibility was highlighted by th ability (CVav% = 4% for buffer and 3% for AU), with an LOD of 16 ± 1 μg L −1 an of 54 ± 2 μg L −1 in buffer solution and an LOD of 22 ± 1 μg L −1 and LOQ of 75 ± AU (Table S1). Notably, these concentrations are below the values of copper as the pathological state of Wilson's diseases [24,25], stimulating the application of to human urine samples.

Copper Quantification via Quenching-Based Bioanalytical Assay
The SEDF fluorescence spectra and dose-response plots in the buffer and in artificial urine (AU) samples are reported in Figure 6. The fluorescence signal was reported as F 0 /F, where F 0 is the fluorescence intensity of the SEDF, and F is the emission signal of the SEDF after the copper addition. The fluorescence intensity at 450 nm after the Cu 2+ addition was plotted versus the Cu 2+ concentration in the range of 0.05-0.5 mM, following a linear trend with remarkable analytical performance (Table S1), both in the buffer and in AU samples, as confirmed by the correlation coefficient R 2 = 0.990 for both and by the linear fitting equations F 0 /F = 4.68 × [Cu 2+ ] + 0.87 used for the Cu 2+ detection in buffer and F 0 /F = 3.38 × [Cu 2+ ] + 0.93 for AU. The excellent assay reproducibility was highlighted by the low variability (CV av % = 4% for buffer and 3% for AU), with an LOD of 16 ± 1 µg L −1 and an LOQ of 54 ± 2 µg L −1 in buffer solution and an LOD of 22 ± 1 µg L −1 and LOQ of 75 ± 3 µg L −1 in AU (Table S1). Notably, these concentrations are below the values of copper associated to the pathological state of Wilson's diseases [24,25], stimulating the application of this assay to human urine samples.  Table S1.

Copper Detection in Human Urine Samples
With the aim to design a bioassay for the urinary Cu 2+ quantification for clin poses, we transferred our experiments in ELISA-type 96-well microplates, allo the immediate and simultaneous analysis of several human urine samples and the turnaround time for the analysis. Another advantage offered by the method veloped is the direct analysis of the urine samples without any pretreatment.
The urine samples were preliminarily assayed by ICP-MS to determine the copper physiological concentration, resulting in the nM range (Table S3). Amo the urine sample n. 3 was selected to be used as a calibrator in the real matrix standard additions (red line in Figure 7), since its Cu 2+ level represents a me within the series. The CVav% (1%) and the slope reported in Table S3 highlight reproducibility and sensitivity of the method, even in the complex real matrix, LOD and LOQ of 59 ± 3 μg L −1 and 97 ± 11 μg L −1 , respectively.
Subsequently, 100 μL of each urine sample was spiked with different kno concentrations (0.05 mM; 0.20 mM, and 0.40 mM, see Table 1) and added to 1800 SEDF solution. These spiked samples were analyzed in parallel by fluorescence MS (Table 1 and Figure 7).
Despite the complexity and the variability of the analyzed urine samples, th clearly highlight the very good accuracy of the assay with respect to the calibra if the urine sample n. 2 showed a slightly higher variability, likely due to the ma bidity and the darker color of the sample. This allows us to foresee the  Table S1.

Copper Detection in Human Urine Samples
With the aim to design a bioassay for the urinary Cu 2+ quantification for clinical purposes, we transferred our experiments in ELISA-type 96-well microplates, allowing for the immediate and simultaneous analysis of several human urine samples and reducing the turnaround time for the analysis. Another advantage offered by the method here developed is the direct analysis of the urine samples without any pretreatment.
The urine samples were preliminarily assayed by ICP-MS to determine the starting copper physiological concentration, resulting in the nM range (Table S3). Among them, the urine sample n. 3 was selected to be used as a calibrator in the real matrix by Cu 2+ standard additions (red line in Figure 7), since its Cu 2+ level represents a mean value within the series. The CV av % (1%) and the slope reported in Table S3 highlight the high reproducibility and sensitivity of the method, even in the complex real matrix, with an LOD and LOQ of 59 ± 3 µg L −1 and 97 ± 11 µg L −1 , respectively.
Subsequently, 100 µL of each urine sample was spiked with different known Cu 2+ concentrations (0.05 mM; 0.20 mM, and 0.40 mM, see Table 1) and added to 1800 µL of the SEDF solution. These spiked samples were analyzed in parallel by fluorescence and ICP-MS (Table 1 and Figure 7). applicability of the method as a diagnostic tool, covering a broad range of Cu 2+ concentrations expected in this type of patient.  Table S2 and were obtained using the piecewise linear fitting implemented in the Originlab software.

Assay Performance Compared to Other Fluorescence-Based Method for Copper Detection
The quenching-based assay here designed was finally compared to the recently developed fluorescence-based methods for Cu 2+ detection in urine [8][9][10][11] (Table S4), i.e., carbon dots (CDs) combined with covalent organic frameworks (COFs), chitosan/L-histidinestabilized silicon nanoparticles (CS/L-His-SiNPs), nitrogen-doped quantum carbon dots (ND-CQDs), and blue CDs (bCDs) combined with green quantum dots (gQDs). These methods offer better LODs values [8,9] (except in Li et al. and Zhang et al., in which the LOD values were not calculated for human urine samples [10,11]). However, these nanostructures require long and laborious synthetic procedures. Contrarily, herein, SEDF was rapidly (2 h) synthesized and, for the first time, this novel biomaterial was applied for diagnostic purposes.

Conclusions
In this study, the fluorescent properties of a serotonin-derived fluorophore were successfully exploited for urinary Cu 2+ determination via a quenching-based bioanalytical method. To the best of our knowledge, this is the first example of a quenching-based assay Com ber Figure 7. Calibration of Cu 2+ 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 F 0 /F at λ em = 450 nm (λ ex = 350 nm) versus the Cu 2+ concentration, where F 0 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. Despite the complexity and the variability of the analyzed urine samples, the results clearly highlight the very good accuracy of the assay with respect to the calibrator, even if the urine sample n. 2 showed a slightly higher variability, likely due to the marked turbidity and the darker color of the sample. This allows us to foresee the realistic applicability of the method as a diagnostic tool, covering a broad range of Cu 2+ concentrations expected in this type of patient.

Assay Performance Compared to Other Fluorescence-Based Method for Copper Detection
The quenching-based assay here designed was finally compared to the recently developed fluorescence-based methods for Cu 2+ detection in urine [8][9][10][11] (Table S4), i.e., carbon dots (CDs) combined with covalent organic frameworks (COFs), chitosan/L-histidinestabilized silicon nanoparticles (CS/L-His-SiNPs), nitrogen-doped quantum carbon dots (ND-CQDs), and blue CDs (bCDs) combined with green quantum dots (gQDs). These methods offer better LODs values [8,9] (except in Li et al. and Zhang et al., in which the LOD values were not calculated for human urine samples [10,11]). However, these nanostructures require long and laborious synthetic procedures. Contrarily, herein, SEDF was rapidly (2 h) synthesized and, for the first time, this novel biomaterial was applied for diagnostic purposes.

Conclusions
In this study, the fluorescent properties of a serotonin-derived fluorophore were successfully exploited for urinary Cu 2+ determination via a quenching-based bioanalytical method. To the best of our knowledge, this is the first example of a quenching-based assay that exploits the fluorescent properties of a polymer derived by an endogenous molecule. The SEDF's spectroscopic features were deeply investigated, and different techniques were applied to characterize this novel material, including mass spectrometry confirming its oligomeric state. The experimental conditions were optimized to obtain the highest emission signal and to improve the assay sensitivity and reproducibility. The whole protocol was performed within 2 h in line with clinical requirements. The Cu 2+ ions were firstly quantified in buffer and in artificial urine obtaining very low variability, high sensitivity, and low LOD and LOQ values. Then, the assay was applied to estimate the Cu 2+ concentration in human urine samples from volunteers and, finally, validated by ICP-MS obtaining very good recovery values and excellent analytical performances in a real matrix, namely, a CV av % of 1%, an LOD of 59 ± 3 µg L −1 , and an LOQ of 97 ± 11 µg L −1 . These values are below the concentrations of urinary Cu 2+ that, for example, determine the pathological state of Wilson's disease (60-240 µg L −1 ), enabling the applicability of the method for Cu 2+ measurement in routine clinical practice without the need of sample pretreatment.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/s23063030/s1, Figure S1: Fluorescence stability over time for serotonin-derivative fluorophore (SEDF); Table S1: Cu 2+ detection in buffer and in AU. Data are from Figure 6; Table S2: Cu 2+ quantification in human urine. Data are from Figure 7; Table S3: Physiological Cu 2+ concentration of human urine samples; Table S4: Fluorescent-based assays for Cu 2+ quantitative analysis in human urine samples.  Informed Consent Statement: Patient consent was waived because the samples consisted of sample residues, normally destined for destruction, used as a biological matrix to disperse the analyte. Furthermore, the residues were anonymized so that no clinical information could be traced back to the sample.