An Organic/Inorganic Hybrid Membrane as a Solid “Turn-On” Fluorescent Chemosensor for Coenzyme A (CoA), Cysteine (Cys), and Glutathione (GSH) in Aqueous Media

Sensors 2012, 12, 2969-2982; doi:10.3390/s120302969 sensors ISSN 1424-8220 www.mdpi.com/journal/sensors Article An Organic/Inorganic Hybrid Membrane as a Solid “Turn-On” Fluorescent Chemosensor for Coenzyme A (CoA), Cysteine (Cys), and Glutathione (GSH) in Aqueous Media Saúl Vallejos, Pedro Estévez, Saturnino Ibeas, Félix C. García, Felipe Serna and José M. García * Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza de Misael Bañuelos s/n, E-09001 Burgos, Spain; E-Mails: [email protected] (S.V.); [email protected] (P.E.); [email protected] (S.I.); [email protected] (F.C.G.); [email protected] (F.S.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +34-947-258-085; Fax: +34-947-258-831. Received: 17 January 2012; in revised form: 23 February 2012 / Accepted: 23 February 2012 / Published: 2 March 2012 Abstract: The preparation of a fluorogenic sensory material for the detection of biomolecules is described. Strategic functionalisation and copolymerisation of a water insoluble organic sensory molecule with hydrophilic comonomers yielded a crosslinked, water-swellable, easy-to-manipulate solid system for water ‘‘dip-in’’ fluorogenic coenzyme A, cysteine, and glutathione detection by means of host-guest interactions. The sensory material was a membrane with gel-like behaviour, which exhibits a change in fluorescence behaviour upon swelling with a water solution of the target molecules. The membrane follows a “turn-on” pattern, which permits the titration of the abovementioned biomolecules. In this way, the water insoluble sensing motif can be exploited in aqueous media. The sensory motif within the membrane is a chemically anchored piperazinedionederivative with a weakly bound Hg(II). The response is caused by the displacement of the cation from the membrane due to a stronger complexation with the biomolecules, thus releasing the fluorescent sensory moieties within the membrane. Keywords: sensory materials; chemosensor; fluorogenic sensor; biomolecules; sensing biomolecules OPEN ACCESS Sensors 2012, 12 2970 1. Introduction The development of sensing molecules for the detection of chemicals is a topic of current interest [1–6]. The recognition of target molecules based on the variation of a macroscopic property of a sensing molecule associated with the specific interactions of the target with the receptor motifs of the sensor can be used to prepare sensory solutions for the easy, cheap and rapid quantification of chemicals by means of a widely used analytical technique (e.g., UV/Vis and/or spectrofluorometry). Moreover, if the receptor and the transducing motifs are chemically bound to a polymer network structure, then the organic material can be described as a solid system, which can potentially be used as a solid kit for the ‘‘dip-in’’ detection of analytes [3]. For medical, biomedical and environmental reasons, biological molecules are among the most important target analytes [7]. Biomolecules containing a thiol group, such as coenzyme A (CoA), L-cysteine (Cys), and glutathione (GSH), play important roles in biological processes including acyl group carrier capability or oxidation/reduction facility (i.e., intramolecular reduction-oxidation metabolic cycles), which occur in hundreds of biochemical reactions [8–12]. The determination of the biomolecule concentration has been undertaken with various methodologies. One of the most interesting is the sensing methodology based on the host-guest supramolecular approach. The supramolecular approach has recently been applied to the determination of Cys [13–27], CoA [8–10], and GSH to a lesser extent [28]. Herein, we describe a sensory organic/inorganic hybrid membrane for the fluorogenic detection of three important biomolecules: CoA, Cys, and GSH (the chemical structures are shown in Scheme 1). The membrane was a dense film consisting of a hydrophilic acrylic network that contained a small amount of piperazinedione-derivative/Hg(II) moieties as the sensory motif toward the biomolecules mentioned above. The piperazinedione-derivative was chemically anchored to the copolymer backbone. The water-swelled membrane responded to the presence of the targets in an aqueous environment at physiological pH with an increase in the fluorescence intensity (i.e., a fluorescence “turn-on” pattern), which permitted the titration of the biomolecules. Scheme 1. Structure of Cys, GSH, and CoA. H2NOH O SH HOOC NNCOOH NH2 OHS HO H Cysteine (Cys) Glutathione (GSH) O OHO POO - OH N NN N NH2 HS NN O O HH O OH CH3 H3C POPO OO O-OCoenzyme A (CoA) Sensors 2012, 12 2971 2. Experimental Section 2.1. Materials The following commercially available materials and solvents were used as received, unless otherwise indicated: mercury(II) acetate (Sigma Aldrich, 98%), glycine (Sigma Aldrich, 99%), methacryloyl chloride (Fluka, 97%), ethylene glycol dimethacrylate (Aldrich, 98%), ethylene glycol (Fluka), acetic anhydride (Sigma Aldrich, puriss.), potassium t-butoxide (Sigma Aldrich, 99.99%), 4-(dimethylamino)benzaldehyde (Sigma Aldrich, 98%), triethylamine (Fluka, 99.5%), lithium chloride (Sigma Aldrich, 99%), 4-nitrobenzaldehyde (Sigma Aldrich, 99%), sodium sulphide nonahydrate (Sigma Aldrich, 98%), dioxane (Probus, 99%), 1-vinyl-2-pyrrolidone (Sigma Aldrich, 99%), N-methyl-2-pyrrolidone (Sigma Aldrich, 99.5%), diethyl ether (VWR, 99.99%), DMSO (Merck, 99%), acetone (Aldrich, 99%), ethanol (Aldrich, 99%), methanol (VWR, for HPLC), DMF (Aldrich, 99%), coenzyme A trilithium salt (Calbiochem, 99.9%), L-glutathione reduced (Alfa Aesar, 97%), and L-cysteine hydrochloride monohydrate (VWR). Azo-bis-isobutyronitrile (AIBN, Fluka, 98%) was recrystallised twice from methanol. 2.2. Measurements 1H and 13C-NMR spectra were recorded in deuterated dimethyl sulphoxide (DMSO-d6) as the solvent using a Varian Inova 400 spectrometer operating at 399.92 and 100.57 MHz, respectively. Infrared spectra (FTIR) were recorded with a Nicolet Impact spectrometer or with a JASCO FT/IT-4100 fitted with a PIKE TECH “Miracle” ATR. Thermogravimetric analysis (TGA) data were recorded using 5 mg of sample under a nitrogen or oxygen atmosphere on a TA Instrument Q50 TGA analyzer at a scan rate of 10 °C min−1. UV-Vis spectra were recorded using a Varian Cary3-Bio UV-Vis spectrophotometer. The fluorescence spectra were recorded using a Varian Cary Eclipse fluorometer. Millipore-Q water was used to prepare the solutions. To determine the tensile properties of the membranes, strips (5 mm in width, 30 mm in length, and 30–45 μm thick) were cut from the polymer films and measured using a Hounsfield H10KM Universal Testing Dynamometer at 20 °C. Mechanical clamps held the sample, and an extension rate of 5 mm min−1 was applied using a gauge length of 10 mm. At least six samples were tested for each polymer, and the data were averaged. 2.3. Intermediates and Monomer Synthesis The overall synthetic steps for the monomer are shown in Scheme 2. Synthesis of 1,4-diacetylpiperazine-2,5-dione (1). Glycine (a total of 100 g, 1.33 mol) was dissolved in ethylene glycol (500 mL) in a 1,000 mL flask fitted with a mechanical stirrer. The mixture was stirred at 170 °C for 3 h, and the solution was cooled at 5 °C for 20 h. The precipitate, piperazine-2,5dione, was collected by filtration and washed with methanol (500 mL). Then, the solid was dissolved in boiling water, and the solution was cooled overnight. The white product was filtered off and washed with methanol. Yield: 30%. M.p.: 330 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 4.64 (2H, s, NH); 3.85 (4H, s, CH2). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 168.46, 43.83. EI-LRMS m/z: Sensors 2012, 12 2972 114 (M+●, 100), 86 (8), 72 (2), 58 (5), 56 (7). FTIR [wavenumbers (cm−1)]: νN-H: broadband (3,250, 2,750); νC=O: 1,696. Piperazine-2,5-dione (18.7 g, 0.164 mmol) and acetic anhydride (85 mL) were added to a 250 mL flask equipped with a reflux condenser. The mixture was stirred at reflux for 7 h. The solvent was removed by distillation. The product 1 was washed with diethyl ether and collected by filtration. Yield: 90%. M.p.: 96 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 4.64 (6H, s, CH3); 3.84 (4H, s, CH2). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 173.61, 168.17, 43.32, 26.12. EI-LRMS m/z: 198 (M+●, 30), 156 (41), 114 (47), 86 (3), 71 (30), 43 (12). FTIR [wavenumbers (cm−1)]: νN-C=O: broadband (3,452, 3,365); νC=O: 1,718. Synthesis of (3Z,6Z)-3-(4-(dimethylamino)benzylidene)-6-(4-nitrobenzylidene) piperazine-2,5-dione (2). A flask equipped with a reflux condenser was charged with 1,4-diacetylpiperazine-2,5-dione (1, 4.94 g, 25 mmol) and 4-(dimethylamino)benzaldehyde (3.72 g, 25 mmol) which were dissolved in DMF (70 mL). Potassium t-butoxide (2.8 g, 25 mmol) was added, and the mixture was stirred at room temperature for 12 h. The product, (Z)-3-(4-(dimethylamino)benzylidene)-1-acetylpiperazine-2,5dione, was precipitated in water and collected by filtration. Finally, the solid was washed with water and methanol. Yield: 50%. M.p.: 210 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 10.19 (1H, s, NH); 7.53 (2H, d, J 8.7, ArH); 6.95 (1H, s, CH); 6.79 (2H, d, J 9.0, ArH); 4.39 (2H, s, CH2); 3.02 (6H, s, CH3); 2.51 (3H, s, CH3). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 172.31, 165.00, 163.19, 151.28, 132.07, 122.99, 122.45, 120.86, 112.39, 46.08, 26.96. EI-LRMS m/z: 287 (M+●, 90), 245 (100), 160 (52), 115 (3), 78 (11), 62 (14). FTIR [wavenumbers (cm−1)]: νN-H: broadband (3,661, 3,310); νC=O: 1,696, 1,597 and 1,521. (Z)-3-(4-(dimethylamino)benzylidene)-1-acetylpiperazine-2,5-dione (2.6 g, 9 mmol) and 4-nitrobenzaldehyde (1.36 g, 9 mmol) were dissolved in DMF (135 mL) in a flask equipped with a reflux condenser. Triethylamine (0.9 g, 9 mmol) was added, and the mixture was stirred at 130 °C for 12 h. A solid was collected by filtration and washed with methanol. Finally, the solid was washed with acetone at its reflux temperature in a flask equipped with a reflux condenser. Yield: 50%. M.p.: not observed (the compound was amorphous; however, an exothermic crystallisation peak was observed at 353 °C). 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 10.43 (1H, s, NH); 10.09 (1H, s, NH); 8.20 (2H, d, J 6.42, ArH); 7.75 (2H, d, J 7.35, ArH); 7.43 (2H, d, J 6.42, ArH); 6.74 (4H, t, J 5.93, ArH); 2.95 (6H, s, CH3). EI-LRMS m/z: 378 (M+●, 100), 332 (3), 287 (1), 215 (1), 159 (37), 117 (5), 89 (4), 77 (1). FTIR [wavenumbers (cm−1)]: νN-H: broadband (3,628, 3,331), νN-H: 3,211; νC=O: 1,674 and 1,626; νNO: 1,578 (asymmetric) and 1,339 (symmetric). Synthesis of N-(4-((1Z)-((Z)-5-(4-(dimethylamino)benzylidene)-3,6-dioxopiperazin-2-ylidene)methyl) phenyl)methacrylamide (3). In a 250 mL flask fitted with a reflux condenser, compound 2 (1.8 g, 4.75 mmol) was dissolved in dioxane (100 mL). Sodium sulphide nonahydrate (3.43 g, 14.25 mmol) was added to the solution, and the mixture was stirred at 80 °C for 24 h. The solution was filtered, and water (400 mL) was added. The resultant precipitate, (3Z,6Z)-3-(4-(dimethylamino) benzylidene)-6-(4aminobenzylidene) piperazine-2,5-dione, was filtered off and washed twice with methanol. Yield: 72%. M.p.: 307 °C. 1 H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 9.81 (2H, s, NH2); 7.47 (2H, d, J 9.03, ArH); 7.32 (2H, d, J 9.03, ArH); 6.78 (2H, d, J 10.08, ArH); 6.69-6.61 (4H, m, ArH); 5.63 (2H, s, NH); 3.00 (6H, s, CH3). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 159.26, 159.12, 150.66, 150.08, 131.66, 131.43, 123.50, 122.82, 121.29, 120.99, 117.10, 116.41, 114.36, 112.60. EI-LRMS Sensors 2012, 12 2973 m/z: 348 (M+●, 100), 334 (2), 306 (1), 218 (1), 161 (17), 159 (22), 133 (24), 131 (10). FTIR [wavenumbers (cm−1)]: νN-H: 3,432, 3,340 and 3,229; νC=O: 1,672 and 1,598. In a 25 mL flask fitted with a reflux condenser and under N2 atmosphere, (3Z,6Z)-3-(4- (dimethylamino)benzylidene)-6-(4-aminobenzylidene) piperazine-2,5-dione (1.2 g, 3.45 mmol) was dissolved in NMP (7 mL). Methacryloyl chloride (0.47 g, 4.5 mmol) was added to the solution, and the mixture was stirred at room temperature for 4 h. An orange solid (monomer 3) was collected by filtration and purified from the crude residue by washing with hot acetone using a Soxhlet apparatus. Yield: 80%. M.p.: 330 °C. 1H-NMR δH (399.9 MHz, DMSO-d6, Me4Si): 10.08 (2H, s, NH); 9.97 (1H, s, NH); 7.79 (2H, d, J 8.7, ArH); 7.56 (2H, d, J 8.7, ArH); 7.48 (2H, d, J 8.7, ArH); 6.77 (4H, m, ArH); 5.86 (1H, s, CH2); 5.58 (1H, s, CH2); 3.01 (6H, s, CH3); 1.99 (3H, s, CH3). 13C-NMR, δC (100.6 MHz, DMSO-d6, Me4Si): 167.77, 159.55, 159.02, 151.09, 141.26, 139.89, 131.89, 130.74, 129.22, 126.63, 123.43, 121.44, 121.14, 120.91, 117.64, 115.12, 112.91, 19.80. EI-LRMS m/z: 416 (M+●, 100), 376 (16), 347 (4), 159 (62), 131 (9), 117 (5), 77 (3). FTIR [wavenumbers (cm−1)]: νN-H: broadband (3,709, 3,100); νC=O: 1,676, 1,624 and 1,595. Scheme 2. Synthesis of the monomer N-(4-((1Z)-((Z)-5-(4-(dimethylamino)benzylidene)- 3,6-dioxopiperazin-2-ylidene)methyl)phenyl)methacrylamide. 2.4. Membrane Preparation Membrane M1 was prepared by the radical polymerisation of a mixture of 1-vinyl-2-pyrrolidone and (3) with a molar ratio of 99.75:0.25. Ethylene glycol dimethacrylate was used as the cross-linking agent (7% mol percentage regarding the overall comonomer molar content), and AIBN (1 wt%) was used as a thermal radical initiator. Membrane M2 was prepared following the same procedure described for the preparation of M1; however, 0.25% molar content of mercury(II) acetate was added (the same concentration of (3)), which resulted in a hybrid organic-inorganic material. The thermal polymerisation was performed in 100 μm thick silanised glass moulds in an oxygen-free atmosphere at 65 °C for 5 h. The structure and the physical appearance are depicted in Figure 1. COOHH2Ni) Ethylene glycol, Δ ii) Ac2O, Δ N NO O O O N N H H O O NO2 N i) Na2S.9H2O, Dioxane, Δ ii) , NMP Cl ONNHH O ON N O H (1) (3) (2) i) , tBuO-K+, DMF NO 2 OHC NOHC ii) , TEA, DMF, Δ Sensors 2012, 12 2974 Figure 1. Chemical structures of the monomers and the copolymer. The copolymer is shown over a digital picture of the sensory film. 3. Results and Discussion 3.1. Material Characterisation Mechanical and thermal resistance are key parameters to determine the suitability of an organic material for technological applications. From a mechanical point of view, M1, a dense membrane, showed good performance. The Young’s modulus was 490 MPa and the elongation at break was 160% at room temperature with a relative humidity of 65%. The hydrophilic membranes were dried at 103 °C for 20 minutes, which resulted in an increase in the Young´s modulus to 1.1 GPa and a decrease in the elongation at break to 12%. The membrane recovered the initial values upon exposure to the ambient atmosphere. The hydrophilic character of the material resulted in a water uptake of 150% upon immersing the membrane in pure water. A comparison between the FTIR spectra of dry M1 and M1 stored in air overnight (under the abovementioned conditions) was performed. The band corresponding to the amide I of the hydrophilic vinylpyrrolidone moieties exhibited a band shift toward lower energy of 18 cm−1 (1,668 to 1,648 cm−1), while the shoulder at 1,727 cm−1 that corresponds to the hydrophobic ester residue of the crosslinker remained unchanged. These observations probably indicate that hydrophilic and hydrophobic microdomains were present in the water-swelled membrane after immersing the membrane in aqueous media for sensing purposes. The polymerisation of the comonomers without and with low molar content mercury (II) acetate (0.25%) (M1 and M2, respectively) resulted in materials with fairly different FTIR spectra (Figure 2). Comparing the spectra of dry samples of M1 and M2 resulted in the observation of an intense band that developed at 1,722 cm−1 for M2, with a concomitant shift of the amide I band to higher energies, from 1,668 to 1,675 cm−1, probably due to the acetate group. monomers R1R2 (3) N N H H O O N N HO NO R1:R 2: OO O O crosslinker film structure monomer molar ratio: M1: X/Y/Z/M = 99.75/0.25/7.00/0.00 M2: X/Y/Z/M = 99.75/0.25/7.00/0.25 Z R2 O R1 O O O O [Hg(II) ] XY M Sensors 2012, 12 2975 Figure 2. FTIR spectra of the dried membranes M1 (black line) and M2 (red line). The thermal resistance of the membranes was evaluated using TGA. The decomposition temperatures that resulted in 5% and 10% weight loss under a nitrogen atmosphere (T5 and T10, respectively) were approximately 360 and 385 °C, which indicates the material had reasonably good thermal stability. M1 and M2 had a first weight loss at 200 °C, which was attributed to the non-reticulated chain ends [29]. The TGA curves of the membranes are shown in Figure 3. The residue remaining after reaching 800 °C was negligible for M1 and approximately 8% for M2, which confirms the influence of the mercury content in the thermal behaviour. The mercury was first oxidised to HgO, which indicates the hybrid nature of the membrane. The immersion of membrane M2 in water resulted in an insignificant loss of bound Hg(II), as determined by comparing the amount of residue that remained at 800 °C under a nitrogen atmosphere for two samples of M2 that were soaked in pure water for 3 and 24 h and subsequently dried. Both samples resulted in a residue of 8%. Nevertheless, the analysis of the role of the Hg(II) by TGA is cumbersome, because of the behaviour of the mercury salts upon heating. Initially, mercury oxides formed, and then, metallic mercury was formed with concomitant sublimation [30]. Changing the atmosphere from nitrogen to air yielded the complete loss of mass at 800 °C for M1 and M2, which gave rise to a zero char yield. Figure 3. TGA curves of membranes M1 and M2. The degradation pattern of M2 after a cycle of soaking in pure water for 3 h with subsequent drying at rt is also included. 4000 3500 3000 2500 2000 1500 1000 50 0 70 75 80 85 90 95 100 νC=O (lactam, amide I) Transmittance (%) Wavenumbers (cm-1) normalized νC=O (ester, crosslinker) 100 200 300 400 500 600 700 800 0 20 40 60 80 100 Weight (%) Temperature (ºC) M2 M2 in water (3h) M1 Sensors 2012, 12 2976 3.2. The Membranes as Sensory Materials The membrane M1 behaves as a sensory material for the fluorogenic detection of Hg(II) in aqueous media. Upon the addition of Hg(II), the fluorescence of the membrane at 548 nm was quenched, which demonstrated that the membrane had “turn-off” fluorescence behaviour in the presence of the cation. This observation was attributed to the interaction of the Hg(II) with the N-terminus of the sensory motif (3) within the membrane at a 1:1 stoichiometry [31]. The integral preparation of a membrane containing equal molar quantities of (3) and Hg(II) (i.e., M2), led to a material with a partially quenched fluorescence. Moreover, fluorescence recovery was observed for M2 upon adding different biomolecules, e.g., CoA, Cys and GSH (see Figure 4). The stronger interaction of these biomolecules with Hg(II) led to a fluorescent chemosensor with fluorescence “turn-on” behaviour, based on the displacement approach [1,2]. A titration curve of the biomolecules was obtained by plotting the fluorescence maxima versus the biomolecule concentration. An illustrative example is shown for CoA in Figure 4. The limit of detection (LOD) was approximately 2 × 10−10 M. Figure 4. Selected fluorescence spectra (left) and titration curve (right) of M2 upon adding increasing quantities of CoA in water at physiological pH (pH = 7.4, TRIS) at an excitation wavelength of 400 nm. The inset for the figure on the right is an expansion of the lower concentrations of the titration curve. 3.3. Copolymer Network/Hg(II) Interaction Prior to the preparation of the membrane M2, the interaction of the monomer containing the sensing motif (3) with Hg(II) was studied in solution. The stoichiometry of the (3):Hg(II) complexes in a DMSO/water solution (90:10) was mathematically determined by analysing the fluorescence quenching process, the intensity maxima variations versus the Hg(II) concentration, and the corresponding Job’s plots. The Job’s plot showed a maximum that appeared at a mole fraction for (3) (χ(3)) of 0.5, which clearly indicated the formation of complexes with a 1:1 stoichiometry, as shown in Table 1 and Figure 5. 530 540 550 560 570 580 590 70 72 74 76 78 80 82 84 86 8.2x10-11 M Fluorescence intensity (a.u.) [CoA] 6.1x10-8 M λ, nm 0.0 2.0x10-8 4.0x10-8 6.0x10-8 0.96 0.97 0.98 0.99 1.00 0.0 1.0x10-9 2.0x10-9 3.0x10-9 4.0x10-9 0.97 0.98 0.99 1.00 I0/I (549 nm) [CoA], M I0/I (549 nm) [CoA], M Sensors 2012, 12 2977 Table 1. Stability constants corresponding to the complex (3):X [X = Hg(II), CoA, Cys and GSH], and CoA:Hg(II). Complex Complex stoichiometry K1 (M−1) K2 (M−1) (3):Hg(II) 1:1 110,000 ± 10,000 – (3):CoA 1:1 22,000 ± 2,000 – (3):Cys 1:1 20,000 ± 8,000 – (3):GSH 1:1 20,000 ± 7,000 – CoA:Hg(II) 1:2 8,400 ± 900 6,000 ± 3,000 Figure 5. Job’s plots corresponding to the following interaction studies: (a) (3) with Hg(II), obtained from fluorescence spectroscopy (591 nm) data corresponding to the titration curve of (3) with mercury cations in DMSO/water (90/10, v/v) at pH = 9.7 (TRIS); and (b) CoA with Hg(II), from UV/Vis spectroscopy (300 nm) data corresponding to the titration of CoA with mercury cations in DMSO/water (90/10, v/v) at pH = 7.4 (TRIS). (a) (b) The strength of the interaction of (3) with Hg(II) in buffered DMSO/water (90/10) solution (pH = 7.4) in terms of the stability constant, K1, corresponding to the host-guest complexes, was analysed by fluorescence spectroscopy. The determination of the 1:1 stoichiometry of the (3):Hg(II) complexes allowed the following equilibrium to be stated: L + M ML K1 (1) which can also be written as the following: ]][[ ][ 1ML ML K= (2) where [L], [M], and [ML] is the equilibrium concentration of (3), Hg(II), and the (3):Hg(II) complex, respectively. From the mass balance and fluorescence data, the following equations can be deduced: CL = [L] + [ML] (3) CM = [M] + [ML] (4) IF = fL[L] + fML[ML] (5) 0.0 0.2 0.4 0.6 0.8 1.0 0 10 20 30 40 (Fo-F)χ(3) χ(3) 0.0 0.2 0.4 0.6 0.8 1.0 0.00 0.05 0.10 0.15 (A-Ao)χCoA χCoA