4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Sigma-Aldrich. FH was synthesized using a modification of a literature method. HEPES solutions were adjusted to pH 8.0 by adding

NaOH (0.1 M) to aqueous HEPES (10 mM). Cationic salts were purchased from Shanghai Experiment Reagent Co., Ltd (Shanhai, China). All the common chemicals used were of analytical grade.

A Mettler Toledo pH meter (Mettler-Toledo International Inc, Switzerland) was used to determined pH. The UVVisible spectra were recorded on a Cary 50 Bio UVVisible spectrophotometer (Agilent, Santa Clara, CA). Fluorescence spectra were measured using a Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA). A PO-120 quartz cuvette (10 mm) was purchased from Huamei Experiment Instrrument Plants (Shanhai, China). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker DRX-300MHz NMR spectrometer (Billerica, MA). A light yellow single crystal of FH was mounted on a glass fiber for data collection. Cell constants and an orientation matrix for data collection were obtained by least-squares refinement of diffraction data from reflections with 2.04˚ - 27.4˚ for FH using a Bruker SMART APEX CCD automatic diffractometer. Data were collected at 173 K using Mo Kα radiation (λ = 0.710713 Å) and the ω-scan technique and corrected for Lorentz and polarization effect (SADABS) [50]. The structures were solved by direct methods (SHELX97) [51], and subsequent difference Fourier map and then refined on F2 using a full-matrix least-squares procedure and anisotropic displacement parameters.

FH was prepared in high yield by reacting fluorescein with hydrazine hydrate in methanol (Scheme 1) according to the literature [52,53]. An excessive hydrazine hydrate (85%, 1.2 mL) was added to a 0.35 g of fluorescein dissolved in 20 ml of ethanol, and the reaction solution was refluxed in oil bath for 8 h. A brown oily product resulted from evacuating ethanol under reduced pressure. The solid product was precipitated by adding water and recrystallized from ethanol/water mixture, producing the fluorescein hydrazide (FH) as a yellow powder with 72% yield (0.25 g). The H₂O/ethanol solution was allowed to evaporate slowly at room temperature for several days and yellow crystals suitable for X-ray crystallography were formed. ¹H NMR ,(DMSO-d₆): δ (ppm) 9.80 (s, 2H), 7.76 (m, 1H), 7.48 (m, 2H), 6.99 (m, 1H), 6.58 (s, 2H), 6.43 (d, 2H), 6.38 (d, 2H), 4.37 (s, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 24.25, 33.00, 113.31, 117.96, 121.58, 121.88, 123.80, 138.69, 156.24, 196.37 (Figure S1(a)); ESI-MS m/z 347.2 [FH+H]⁺ (calcd. 347.l) (Figure S1(b)); Elemental analysis (calcd.%) for C₂₀H₁₄N₂O₄: C, 69.36; N, 8.09; H, 4.07: Found: C, 69.30; N, 8.11; H, 4.01. Crystal data for C₂₀H₁₆N₂O₅ (Figure S1(c)): crystal size: 0.22 × 0.2 × 0.1, triclinic, space group P-1 (No. 2). a = 7.5959(15) Å, b = 10.690(2) Å, c = 11.028(2) Å, α = 104.34(3)˚, β = 109.09(3)˚, γ = 99.67(3)˚, V = 789.0(4) ų, Z = 2, T = 173K, θ_max  = 25.0˚, 7521 reflections measured, 2762 unique (R_int = 0.0412). Final residual for 250 parameters and 2503 reflections with I > 2σ(I): R₁ = 0.0622, wR₂ = 0.1390 and GOF = 1.17.

Since the chemosensor was not fully soluble in 100% aqueous media, ethanol was used as a solubilizing medium. FH stock solutions were prepared in ethanol. The UV-Vis and fluorescence spectra were obtained in mixed ethanol /HEPES aqueous buffer (1:1, v/v, 10 mM, pH 8.0) solution. Aqueous metal ion solutions were also prepared. Fluorescence measurements were carried out with a slit width of 10 nm.

The UV-Vis spectrum was characterized by a main band centred at 641 nm. The low detection threshold for Hg²⁺ was in the order of 10⁻⁶ - 10⁻⁵ M and at this level the colour change was very obvious. The fluorescence emission was measured for each sample by exciting at 450 nm and spectra and measuring from 475 - 700 nm. The sensitivity range for Hg²⁺ was 10⁻⁷ - 10⁻⁶ M.

The complexation ability of FH with Hg²⁺ ion was investigated by UV-Vis absorption techniques. FH does not absorb in the range of 400 - 800 nm in a mixed solution of ethanol/HEPES (v/v = 1:1). Figure 2 shows the spectral changes of FH in ethanol/HEPES (v/v = 1:1) upon addition of various competitive metal ions, such as Hg²⁺,

Mg²⁺, Ca²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Ni²⁺, Bi³⁺, Co²⁺, VO²⁺, Mn²⁺, Ba²⁺, Cd²⁺, Pb²⁺, Sn²⁺, Yb³⁺, Cr³⁺, La³⁺, Er³⁺, etc. From UV/Vis spectra (Figure 2(a)), we can clearly observe a new absorption band centered at 397, 504 and 641 nm for FH (30 µM) in the presence of 1 equiv of Hg²⁺. In contrast, other ions lead to almost no spectral changes (Figures 2(a) and (b)). In our present experiments, HgCl₂ as a Hg²⁺ source was gradually added to the ethanol /HEPES (v/v = 1:1) FH solution. Notably, the new band at 397, 504 and 641 nm appeared and concomitantly grew with increasing Hg²⁺ concentration (Figure 2(c)).

The ability of FH to selectively sense Hg²⁺ was determined analysis of the fluorescence spectra obtained with 1 μM of FH in ethanol/HEPES (v/v = 1:1) in the presence of a number of cations including Mg²⁺, Ca²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Ni²⁺, Bi³⁺, Co²⁺, VO²⁺, Mn²⁺, Ba²⁺, Cd²⁺, Pb²⁺, Sn²⁺, Yb³⁺, Cr³⁺, La³⁺ and Er³⁺ etc. (100 equiv to Hg²⁺, respectively). The fluorescence spectra (Figure 3) show a similar result, which is consistent with that of UVVisible spectra. Addition of 3 equiv of Hg²⁺ ion results in an obviously enhanced fluorescence at 522 nm (OFFON), with an excitation at 460 nm while other ions induce no increase in fluorescence (Figure 3(a)). More interestingly, Hg²⁺-induced fluorescence-on change for the FH is visual with a solution color change from colorless to green under illumination with a 365 nm UV lamp (Figure 3(b)). As shown in Figure 3(c), a new emission band peak appears with the fluorescence intensity increasing with increase in Hg²⁺ concentration. Both UVVis and fluorescence results indicate that FH shows a good selectivity and sensitivity toward Hg²⁺ over other competitive cations.

Furthermore, a plot of fluorescence intensity when FH is titrated with 3 µM of Hg²⁺ shows good linearity (correlation coefficient of R = 0.9985) for a Hg²⁺ concentration range of 0.25 - 3 µM (Figure 4).

The above-mentioned UV-Visible light absorption occurred at a pH of 8.0, which is close to physiological conditions. At a pH of 9.0, it seems that Hg²⁺ detection is possible, and that the absorbance was affected by solution alkalinity. At all other pH conditions, no notable change in either color or UV-Visible spectrum was noted (Figure 5).

To provide reasonable envidence of FH sensing of Hg²⁺ ion, electrospray ionization mass spectrometry (ESI-MS) analysis was conducted (Figure S2). Mass peaks at m/z 531.2 corresponding to [Fluorescein + Hg]⁺ are clearly

observed, This provides direct evidence for the proposed response mechanism (Scheme 2). The hydrolysis complex with fluorescein anion is responsible for the above dual color and fluorescence changes.