A polymeric sensor for the chromogenic and luminescent detection of anions

European Polymer Journal 45 (2009) 272–277

Contents lists available at ScienceDirect

European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

A polymeric sensor for the chromogenic and luminescent detection of anions Narinder Singh, Navneet Kaur, John Dunn, Ruth Behan, Ray C. Mulrooney, John F. Callan * School of Pharmacy and Life Sciences, The Robert Gordon University, Aberdeen, Scotland AB10 1FR, UK

a r t i c l e

i n f o

Article history: Received 19 August 2008 Received in revised form 16 October 2008 Accepted 23 October 2008 Available online 7 November 2008

Keywords: Fluorescence Co-operation binding Fluoride Polysiloxane

a b s t r a c t A polysiloxane based thiourea coupled sensor has been developed for the determination of anions from changes in the UV–Vis or fluorescence spectra. A comparative account of the photophysical properties of the monomer and polymer units bearing the thiourea moiety revealed better fluoride recognition in the polymeric framework. The fluoride recognition by the polymeric sensor was attributed to co-operative binding involving a DMSO molecule and a fluoride ion between thiourea groups on adjacent residues. The polymeric sensor can measure fluoride at two different concentration ranges by using either absorbance or emission signalling. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Anions play key roles in a number of important physiological and environmental processes [1]. Consequently, the development of chromogenic and luminescent sensors to accurately detect anions is of great current interest [2]. The ionophore component present in these sensors varies greatly and can consist of positively charged units such as guanidinium groups [1a] or charge neutral ureas and thioureas [3] among others. Ditopic sensors have shown improvements in selectivity/sensitivity over their monotopic counterparts by providing an additional binding site to assist with ion chelation [4]. We have also shown that by incorporating ion receptors onto the surface of a preformed CdSe/ZnS core-shell quantum dot, the binding affinity of the receptor was modified [5]. As an extension to this, the incorporation of receptors on a polymeric framework may lead to a macromolecular assembly that can provide multiple binding sites. Here, we graft a known fluorescent anion sensor onto a polysiloxane using the thiolene reaction. Specifically, 1-[2-(2-allyl-1,3-dioxo-2,3* Corresponding author. E-mail address: [email protected] (J.F. Callan). 0014-3057/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2008.10.040

dihydro-1H-benzo [de]isoquinolin-6-ylamino)-ethyl]-3phenyl-thiourea (3) was grafted onto poly(mercaptopropylmethyl)siloxane (PMPMS) to furnish a side chain anion sensing polymer (4). The monomer 3 was almost structurally identical to that used by Gunnlaugsson et al. as a fluoride sensor, the only difference being the vinylic unit present in 3 [3e]. The monomer was designed according to the fluorophore-spacer-receptor format of photoinduced electron transfer (PET) based sensors [6]. The napthalamide fluorophore can absorb and emit photons in the visible region of the electromagnetic spectrum and so is useful when considering intracellular applications. The spacer, an ethyl unit, functions to keep the fluorophore and the charge neutral thiourea receptor close enough so that electron transfer between them is possible, but also far enough away so that they do not directly influence each other in the excited state. Therefore, upon binding a target analyte, the reduction potential of the receptor is raised which increases the rate of PET from the receptor to the fluorophore and fluorescence is quenched. PMPMS was chosen as polymer as polysiloxanes typically have low glass transition temperatures, low viscosity at room temperature and are generally biocompatible. The selectivity and sensitivity of the new polymeric sensor was

N. Singh et al. / European Polymer Journal 45 (2009) 272–277

determined against a range of anions and the results compared directly with its monomeric analogue. 2. Experimental 2.1. General PMPMS was purchased from Fluorochem (6.6 kDa). All other reagents used were of the highest grade obtainable and were purchased from Aldrich. Absorbance measurements were recorded on an Agilent UV–Vis spectrometer using 10 mm quartz cuvettes. Fluorescence measurements were recorded on a Perkin-Elmer LS55 luminescence spectrometer using 10 mm quartz cuvettes. Excitation slit size was 10 nm and emission slit size was 10 nm. Scan speed was set at 500. NMR spectra were recorded on a Bruker AVANCE 400 MHz spectrometer. Chemical shifts are reported in parts per million, downfield of TMS. Accurate Mass data was provided by the EPSRC National Mass Spectrometry Service, UK. 2.1.1. Synthesis of 2-allyl-6-bromo-benzo[de]isoquinoline1,3-dione (1) The synthesis of 1 was performed following the literature procedure [7a]. 2.1.2. Synthesis of 2-allyl-6-(2-amino-ethylamino)benzo[de]isoquinoline-1,3-dione (2) Synthesis of 2 has been prepared before but was not characterised fully [7b]. We used a different method to prepare 2:1 (4.02 g, 12.72 mmol) was dissolved in an excess of ethylene diamine (10 mL) and heated at 80 °C for 18 h. The reaction mixture was poured slowly into water and the resulting precipitate collected by filtration. The product was dried in vacuo to yield the product as a yellow solid (2.92 g, 77.80%). Mp 155-158 °C dH (400 MHz, CDCl3), 8.53 (1H, d, J = 7.2 Hz, Naph-H) 8.41 (1H, d, J = 8.4 Hz, Naph-H) 8.12 (1H, d, J = 8.0 Hz, Naph-H) 7.56 (1H, t, J = 7.8 Hz, Naph-H) 6.65 (1H, d, J = 8.0 Hz, Naph-H) 6.13 (1H, br s, NH) 5.94 (1H, m, CH), 5.23 (1H, d, Jac = 17.0, Jbc = 1.6 Hz, @CH) 5.10 (1H, d, Jab = 10.0, Jbc = 1.6 Hz, @CH) 4.72 (2H, d, J = 5.6 Hz, @CACH2) 3.35 (2H, t, J = 5.6 Hz, ANCH2) 3.12 (2H, t, J = 5.6 Hz, ANCH2). dC (400 MHz, CDCl3) 164.5, 163.9, 149.8, 134.6, 132.7, 131.2, 129.9, 126.4, 124.7, 122.9, 120.5, 117.0, 110.1, 104.4, 44.9, 42.1, 40.1 HRMS (ES+): (M+H) calculated for C17H17O2N3, 296.1394; found 296.1391. 2.1.3. Synthesis of 1-[2-(2-allyl-1,3-dioxo-2,3-dihydro-1Hbenzo [de]isoquinolin-6-ylamino)-ethyl]-3-phenyl-thiourea (3) To a solution of 2 (2.00 g, 6.80 mmol) in DMF (100 ml) was added phenylisothiocyanate (0.87 g, 6.42 mmol). The solution was stirred overnight at room temperature. Excess solvent was removed under vacuum affording a yellow/orange solid which was stirred in cold chloroform, filtered and dried to yield the product as a yellow powder. (1.55 g, 53.10%) m.p. 218–220 °C, dH (400 MHz, DMSOd6), 9.76 (1H, s, NHurea) 8.77 (1H, d, J = 8.0 Hz, Naph-H) 8.52 (1H, d, J = 7.4 Hz, Naph-H7) 8.34 (1H, d, J = 8.4 Hz,

273

Naph-H) 7.99 (1H, br s, NHurea) 7.89 (1H, br s, NH) 7.78 (1H, t, J = 8.0 Hz, Naph-H) 7.38 (4H, m, 4  Ar-H) 7.19 (1H, m, Ar-H) 7.05 (1H, d, J = 8.4 Hz, Naph-H) 5.99 (1H, m, CH@) 5.17 (1H, s, @CH) 5.13 (1H, d, J @ 5.8 Hz, @CH) 4.70 (2H, d, J = 5.2 Hz, AC@CH2) 3.92 (2H, q, J = 6.0 Hz, NHCH2) 3.69 (2H, q, J = 5.8 Hz, NHCH2) dC (400 MHz, DMSO-d6) 180.5, 163.2, 162.4, 150.5, 138.7, 134.0, 133.3, 130.5, 129.4, 128.8, 128.7, 124.4, 124.2, 123.5, 121.7, 120.1, 116.0, 107.7, 103.9, 79.1, 42.2, 41.3, 35.7, 30.7 HRMS calcd for C24H22N4O2S 431.1356 (M+H)+ found 431.1356. 2.1.4. Synthesis of 4 PMPMS (87.8 mg, 0.65 mmol), 3 (280 mg, 0.65 mmol of repeat units) AIBN (0.05 mg, catalytic amount) and DMF (10 mL) were mixed in a tube flushed with nitrogen. The tube was sealed and placed in an oven at 50 °C for 48 h. The contents were poured onto acidified methanol and the product isolated by filtration. The product, a yellow solid was re-dissolved in DMF and re-precipitated from acidified methanol to yield the final product which was filtered and dried in vacuo (160 mg). 3. Results and discussion The synthesis of monomer 3 is shown in Scheme 1. It was prepared from 4-bromo-1,8-napthalic anhydride in three steps following known procedures [7]. It was then grafted onto PMPMS at one molar equivalence using the thiolene reaction to produce polymer sensor 4. Successful grafting was confirmed by 1H NMR spectroscopy with Fig. 1 showing the stacked NMR spectra of 3, 4 and PMPMS in descending order. The olefinic protons present in 3 at 5.95 and 5.15 ppm were absent in the spectrum of 4. There was also an upfield shift of the signal at 4.65 ppm in 3, reflecting the methylene protons adjacent to the olefinic group, to 3.95 ppm in 4. In addition, the thiol signal present at 1.25 ppm in PMPMS was significantly reduced in 4, also indicating a substantial graft of the thiol groups with 3. New broad signals observed at 3.95, 3.50, 2.60, 1.75 and 1.60 reflect the newly created and existing methylene protons that flank either side of the thioether sulfur atom. Although the peaks were quite broad, we estimated the grafting efficiency to be 87 ± 5% by relative integration. Based on this result the molecular weight of 4 was calculated to be 15.0 ± 0.75 kDa IR analysis also showed that the peak at 2558 cm 1 in PMPMS, reflecting the S–H stretch, was absent in the spectrum of 4. The UV–Vis spectra of 3 and 4 recorded in DMSO are shown in Fig. 2. Both exhibited two main bands, the first at kmax 270 nm being due to the phenyl chromophore and the second at kmax 444 nm due to the internal charge transfer (ICT) excited state of the napthalamide moiety. The ground state properties of 3 and 4 were also determined in the presence of the tetrabutylammonium salts of fluoride, chloride, bromide, acetate, hydrogensulfate and dihydrogenphosphate. No major changes were observed for 3 with any of the anions. In contrast, a similar compound prepared by Gunlaugsson et al. showed increases in absorbance for the shorter wavelength band attributed to the phenyl component of the thiourea recep-

274

N. Singh et al. / European Polymer Journal 45 (2009) 272–277

Si O O

O

O

O

(i)

Br

N

O

(ii)

O

Br

N

O

O

N

O

(iii)

O

2

n

N

O

NH

NH 2 *

S

NH

NH

1 Si O

(iv)

n

HN

S NH

3

SH

NH

PMPMS

HN

S

4 Scheme 1. Synthesis of 1–4. (i) Allyl amine, DMF, 50 °C, 18 h (ii) ethylene diamine, 80 °C, 18 h (iii) phenylisothiocyanate, DMF, 25 °C, 18 h. (iv) PMPMS, DMF, AIBN, 50 °C, 48 h.

a

H2O

DMSO

a 0.8 0.7

b

Absorbance

0.6 0.5

[ F-] = 9.0 mM

0.4 0.3 0.2

[ F -] = 0 mM

0.1 0 260

c

310

360

410 460 Wavelength (nm)

510

560

b 1.4 Fig. 1. 1H NMR spectra of (a) 3 (b) 4 and (c) PMPMS. Solvent = d6-DMSO for (a) and (b) and CDCl3 for (c).

tor [3e]. As this chromophore is directly connected to the thiourea receptor it was expected that we would also see changes in this band for 3 but surprisingly these were absent. In the case of 4 substantial changes were observed upon the addition of fluoride with the other ions having virtually no effect. In fact the intensity of the absorbance of both bands was observed to increase substantially with increasing concentration of fluoride. As described earlier, this was expected for the shorter wavelength band as the chromophore is directly connected to the receptor. However, the reason for the enhancement of the ICT band was not expected as the ethyl spacer should prevent any significant ground state interactions between the thiourea and napthalimide units. Several groups have previously demonstrated using di- or polypodal anion sensors containing urea or 2-aminobenzamidazole receptors that DMSO may

Absorbance

1.2 1 0.8

[ F -] = 9.0 mM

0.6 0.4

[ F-] = 0 mM

0.2 0 260

310

360

41 460 410 Wavelength (nm)

510

560

Fig. 2. UV–Vis titration of (a) 3 and (b) 4 with tetrabutylammonium fluoride in DMSO. [3] = 3.4  10 5 M; [4] = 9.5  10 5 M.

assist in the co-ordination of anions between adjacent pods [8]. To determine if DMSO could assist 4 in the binding of fluoride we repeated the titration in 100% acetonitrile. As shown in Fig. 3a, no significant enhancement was observed for either band, the only difference being a slight red shift in the band at 450 nm, most likely caused by the deprotonation of the thiourea protons at high pH [3e]. Furthermore, when 4 was titrated with tet-

275

N. Singh et al. / European Polymer Journal 45 (2009) 272–277

a

0.3

Absorbance

0.25 0.2 0.15 0.1 0.05 0 260

310

360

410 460 Wavelength (nm)

N

OO

510

560

b O

N

NH

HN H N

N H S

N H

O

F-

O

S

H N

S

Fig. 3. (a) Titration of 4 with tetrabutylammonium fluoride in CH3CN. [4] = 9.5  10 5 M [F-] = 0 ? 9.0 mM. (b) Illustration of how DMSO may co-operate in the binding of a fluoride ion between adjacent residues of 4.

Unfortunately, we were not able to confirm this hypothesis by 1H NMR due to the broadness of the peaks. Nevertheless, sigmoidal binding profiles were obtained by plotting changes of absorbance at both 270 and 444 nm against fluoride concentration (Fig. 5). In fact both plots exhibited very similar binding profiles. Using the equation: log [Amax A]/[A Amin] = log [anion] log b (where Amax and Amin are the maximum and minimum absorbance and A is the measured absorbance) the binding constants, log b, were calculated as 2.59 at 270 nm and 2.61 at 450 nm (Table 1) [9]. When 3 and 4 were excited at 450 nm both compounds exhibited a broad emission with kmax 540 nm, characteristic of the napthalimide fluorophore. No differences in terms of peak position or shape was observed between the two, however the quantum yield of 3 (/F = 0.41) was significantly greater than 4 (/F = 0.12). This could be due to conformational reasons with the polymer adopting a conformation that alters the polarity of the local environment of the fluorophore [10]. However, due to the ICT nature of the napthalimide excited state, such a change in polarity would be expected to manifest itself as a change in the emission kmax, which was not observed (see Fig. 7). Another possibility is that there may be a PET quenching contribution from neighboring thiourea receptors to a single fluorophore resulting in an increased residual PET for 4 even in the unbound state. The effect of anions on the quantum yield of both 3 and 4 was investigated in DMSO. Addition of chloride, bromide and hydrogensulfate had no major effect on the intensity of

a

1.2 1.1 1

0

2

4

6

8

10

-

[F ] mM

A / Ao (@ 270 nm)

Absorbance

1.2 1 0.8 0.6 0.4 0.2 260

1.3

b 1.4

1.4

0

1.4 A / Ao (@ 444nm)

rabutylammonium hydroxide, the UV profile was much different to that with fluoride, with the appearance of a new red-shifted band at kmax 538 nm due to napthalimide NH deprotonation (Fig. 4) [3e]. This suggests that there is no interaction between fluoride and the acidic napthalimide NH proton and that DMSO must participate somehow in the binding of fluoride by 4, most likely by facilitating the association of receptors in adjacent residues of the polymer, as depicted in Fig. 3b. This may lead to an aggregation of the napthalimide groups resulting in an enhancement of the absorbance at 444 nm.

1.5

1.3

1.2

1.1

1 310

360

410 460 Wavelength (nm)

510

560

Fig. 4. Absorbance spectra of spectra of 4 upon increasing amounts of tert-butyl ammonium hydroxide. Solvent = DMSO; [4] = 9.5  10 5 M.

0

2

4

6

8

10

-

[F - ] mM Fig. 5. Plot of relative absorbance against concentration of fluoride ion for 4 measured at (a) 444 nm and (b) 270 nm. Solvent = DMSO; [4] = 9.5  10 5 M.

276

N. Singh et al. / European Polymer Journal 45 (2009) 272–277

Table 1 Photophysical properties of 3 and 4.

3 4 3 + F3 + Cl3 + Br3 + AcO3 + HSO4 3 + H2PO4 4+F b 4 + Cl 4 + Br4 + AcO 4 + HSO4 4 + H2PO4 a

kmax (UV) nm

kmax (EM) nm

emax (mol

444 440 446 444 444 446 444 448 441 440 440 444 441 443

525 525 523 525 525 524 525 525 524 524 525 525 524 525

4.18 3.75 – – – – – – – – – – – –

1

dm3 cm

1

)

UFLU a

% F Red

log b

0.41 0.12 0.15 0.36 0.36 0.20 0.33 0.31 0.06 0.11 0.11 0.07 0.10 0.07

– – 62 12 12 40 20 25 53 12 12 42 22 44

– – 1.92 – – 2.55 – 2.07 2.59 (UV); 2.61 (UV); 1.90 (Flu); 4.43 (Flu) – – 3.10 – 2.98

Quantum Yields calculated with reference to fluorescein. Two binding constants were obtained from UV–Vis and fluorescence for fluoride.

b

1000 900 800 700

Intensity

the emission of 3 or 4. Addition of acetate resulted in an approximate 40% quenching of the original intensity for both 3 and 4. The addition of fluoride and dihydrogenphosphate also resulted in quenches of the original intensity, however the profile and magnitude was different for 3 compared to 4. For 3, (Fig. 6a) there was clear evidence of a single binding event with a large quench in intensity observed at high concentrations of fluoride (log b = 1.92) [11]. For 4, there was evidence of two distinct binding

[F-] = 0 mM

600 500 400 300

[F-] = 70 mM

200 100 0

a

1

580 Wavelength

630

680

0.6 0.4 0.2

0.9

0.7

3

-

5

- log [F]

0

0

2

4

6

4

6

-

- log [F ]

b Relative Intensity

530

Fig. 7. Fluorescence spectra for 4 upon addition of increasing amount of fluoride in DMSO. [4] = 5.0  10 6 M.

Relative Intensity

Relative Intensity

0.8

480

1 0.8 0.6 0.4 0.2 0

0

2

- log [H2PO4- ] Fig. 6. Plot of relative fluorescence intensity against concentration for (a) 3 (filled circles) and 4 (open circles) titrated with fluoride (inset shows an expansion of the lower concentration range) and (b) 3 (filled triangles) and 4 (open triangles) titrated with dihydrogenphosphate. Solvent = DMSO; [3] = 2.3  10 7 M; [4] = 5.0  10 6 M.

events. The first resulted in only a partial quench of fluorescence (20%) and happened relatively low concentrations of fluoride (log b = 4.43). The second was almost identical to that for 3 with a binding constant, log b of 1.90, although the final relative intensity was higher for 4 at 47% compared to 38% for 3 which was somewhat lower than that observed by Duke and Gunnlaugsson (Table 1) [3e]. The quenching of fluorescence at lower concentrations of fluoride may again be due to co-operative binding between DMSO and fluoride in adjacent residues as shown in Fig. 3. At higher concentrations of fluoride the interaction between it and the polymer resembles that of the monomer 3 and the second binding event occurs with a similar binding constant to that of the monomer. A marked difference was also observed in the fluorescence binding profiles of 3 and 4 upon addition of dihydrogenphosphate. Fig. 6b shows a plot of relative intensity against concentration of dihydrogenphosphate for 3 and 4. A much more pronounced quench was observed for 4 compared to 3 with a 25% reduction for 3 and 44% for 4. The binding constant was also greater for 4 (log b = 2.98) than for 3 (log b = 2.07). This is similar to that observed for fluoride but without the appearance of two binding events. In all cases the quenching observed upon addition of anions is due to an increase in the reduction potential

N. Singh et al. / European Polymer Journal 45 (2009) 272–277

of the receptor upon binding resulting in a more effective PET process. In conclusion, we have shown that by incorporating a thiourea receptor onto the side chain of polysiloxane the binding profile against certain anions in DMSO was altered. Significantly, we were able to monitor the concentration of fluoride at two different concentration levels by switching between UV–Vis and fluorescence. We believe that DMSO assists the binding of fluoride between adjacent thiourea residues. This offers the possibility of processing these polymers into thin films with potential use in diagnostics. To the best of our knowledge, this is the first reported example of a dual chromogenic/fluorescent sensor for anions.

[4]

[5] [6]

Acknowledgements The authors thank the EPSRC and RGU for financial assistance. We also thank the EPSRC national Mass Spec service for accurate mass data. References [1] [a] Martínez-Máñez R, Sancenón F. Chem Rev 2003;103:4419; [b] Suksai C, Tuntulanti T. Chem Soc Rev 2003;32:192. [2] [a] Yocum CF. Coord Chem Rev 2008;252:296; [b] Lenthall JT, Steed JW. Coord Chem Rev 2007;251:1747; [c] Amendola U, Esteban-Gomez D, Fabbrizzi L, Licchelli M. Acc Chem Res 2006;39:343; [d] Gale PA. Acc Chem Res 2006;39:465; [e] Katayev EA, Ustynyuk YA, Sessler JL. Coord Chem Rev 2006;250:3004; [f] Davis AP. Coord Chem Rev 2006;250:2939; [g] Schmidtchen FP. Coord Chem Rev 2006;250:2918. [3] [a] Evans LS, Gale PA, Light ME, Quesada R. Chem Commun 2006:965; [b] Pfeffer FM, Seter M, Lewcenko N, Barnett NW. Tetrahedron Lett

[7]

[8]

[9] [10]

[11]

277

2006;47:5241; [c] Pfeffer FM, Buschgens AM, Barnett NW, Gunnlaugsson T, Kruger PE. Tetrahedron Lett 2005;46:6579; [d] Gunnlaugsson T, Davis AP, Hussey GM, Tierney J, Glynn M. Org Biomol Chem 2004;2:1856; [e] Duke RM, Gunnlaugsson T. Tetrahedron Lett 2007;48:8043. [a] Gunnlaugsson T, Davis AP, O’Brien JE, Glynn M. Org Biomol Chem 2005;3:48; [b] Singh N, Jang DO. Org Lett 2007;9:1991; [c] Singh N, Lee GW, Jang DO. Tetrahedron 2008;64:482. Singh N, Mulrooney RC, Kaur N, Callan JF. Chem Commun 2008:4900. [a] Tsien RY. Am J Physiol 1992;263:C723; [b] Bissell RA, de Silva AP, Gunaratne HQN, Lynch PLM, Maguire GEM, McCoy CP, Sandanayake KRAS. Top Curr Chem 1993;168:223; [c] Czarnik AW. Adv Supramol Chem 1993;3:131; [d] Czarnik AW. Acc Chem Res 1994;27:302; [e] Fabbrizzi L, Poggi A. Chem Soc Rev 1995;24:197; [f] de Silva AP, Gunaratne HQN, Gunnlaugsson T, Huxley AJM, McCoy CP, Rademacher JT, Rice TE. Chem Rev 1997;97:1515; [g] Fabbrizzi L, Licchelli M, Pallavicini P. Acc Chem Res 1999;32:846; [h] Fabbrizzi L. Coord Chem Rev 2000;205:1; [i] Kojim H, Nagano T. Adv Mater 2000;12:763; [j] Rurack K, Resch-Genger U. Chem Soc Rev 2002;31:116; [k] de Silva AP, McClean GD, Moody TS, Weir SM. In: Nalwa HS, editor. Handbook of photochemistry and photobiology. CA: American Scientific Publishers, Stevenson Ranch; 2003. 217.; [l] Callan JF, de Silva AP, Magri DC. Tetrahedron 2005;61:8551. [a] Bojinov V, Konstantinova T. Dyes Pigments 2002;54:239; [b] Lu ZJ, Wang PN, Zhang Y, Chen JY, Zhen S, Leng B, et al. Anal Chim Acta 2007;597:306. Moon KS, Singh N, Lee GW, Jang DO. Tetrahedron 2007;63:9106; Burns DH, Calderon-Kawasaki K, Kularatne S. J Org Chem 2005;70:2803. Magri DC, Callan JF, de Silva AP, Fox DB, McClenaghan ND, Sandanayake KRAS. J Fluoresc 2005;15:769. Poteau X, Brown AI, Brown RG, Holmes C, Matthew D. Dyes Pigments 2000;47:91; Uchiyama S, Matsumura Y, de Silva AP, Iwai K. Anal Chem 2003;75:5926. Binding constants were obtained from the equation – log (FMAX F)/ (F FMIN) = log [Anion] + log b where FMAX is the maximum fluorescence intensity, FMIN is the minimum fluorescence intensity and F the observed fluorescence intensity. See Ref. [9].