Transition metal induced switch of fluorescence and absorption response of a Zn(II)porphyrin–DNA conjugate to cysteine derivatives

Abstract

We report a supramolecular zinc(II)tetraarylporphyrin–oligothymidine/metal ion system (ZnPorT8/M²⁺) for dual optical sensing of cysteine (Cys) and glutathione (GSH) with a tunable spectroscopic outcome. Transition metal ions (M²⁺ = Hg²⁺ or Cu²⁺) allow the switching of the emission and absorption response of the ZnPorT8/M²⁺ complex. The ZnPorT8/Hg²⁺ complex exhibits turn-on fluorescence sensing (fluorescence enhancement), whilst the ZnPorT8/Cu²⁺ system exhibits turn-off sensing (emission quenching) of Cys and GSH. The ZnPorT8/Hg²⁺ and ZnPorT8/Cu²⁺ complexes have an excellent limit of detection (LOD(em)) for Cys (5.95 nM and 11.99 nM, respectively) and GSH (3.34 nM and 13.50 nM, respectively).

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Introduction

Porphyrins and metalloporphyrins have found widespread use as recognition and/or reporting components in many diverse sensing systems due to their modular electronic and structural properties.^1–6 They have been successfully used for fluorescence mapping of viscosity,^7,8 electrochemical detection of explosives,⁹ chiroptical sensing of left-handed Z-DNA,^10–13 phosphorescent oxygen sensing,¹⁴ emission detection of Cu(II),¹⁵ amperometric sensing of organophosphate pesticides,¹⁶ or optical determination of bacterial exosporium sugars.¹⁷ We have recently reported highly sensitive optical detection of Hg²⁺ by the zinc(II)porphyrin–oligothymidine bioconjugate ZnPorT8 (Fig. 1).¹⁸ The addition of Hg(II) and Cu(II) lead to complete and partial emission quenching, respectively. The quenching was very likely caused by photoinduced electron transfer upon binding of mercury(II) to trispyridylphenyl-porphyrin. Mercury and copper ions exhibit a very high affinity for the thiol group and form stable complexes with cysteine.^19,20 Interaction between Cys and Hg(II) has been utilized for turn-on^21–28 or turn-off^29,30 emission sensing of Cys by multicomponent supramolecular systems with excellent detection limits ranging from 2.0 to 600 nM. Similarly, Cu-complexes have been explored for spectrophotometric Cys detection.^31–34 Detection of Cys and its derivative glutathione (GSH) is of high importance for diagnosing biothiol related health problems,^35–39 since Cys and GSH are crucial in maintaining the physiological redox homeostasis in biological systems.^40–44 Optical sensing (e.g. fluorescence and absorption) is especially attractive since it is non-destructive, inexpensive, and allows high throughput real time detection without the need for derivatization.^45–47 Herein we report a supramolecular system assembled of ZnPorT8 and a transition metal ion (either Hg(II) or Cu(II)) for highly sensitive and selective optical sensing of Cys and GSH in water. Importantly, transition metal ions allow switching the emission and absorption recognition response of the ZnPorT8/metal complex. The ZnPorT8/Hg²⁺ complex offers a turn-on fluorescence sensing (fluorescence enhancement), while the ZnPorT8/Cu²⁺ system gives reversed turn-off sensing (emission quenching) of biothiols.

Fig. 1 Structure of Zn(II)tetraarylporphyrin–octathymine conjugate ZnPorT8.

Results and discussion

ZnPorT8/Hg²⁺ system: Cys and GSH optical sensing

We have shown that the fluorescence of ZnPorT8 could be significantly decreased (up to 94.2% signal reduction) by Hg²⁺ ions upon their binding to trispyridylphenyl-porphyrin. Since Hg²⁺ ion also forms highly stable complexes with cysteine,⁴⁸ we have decided to explore biothiols optical recognition with supramolecular ZnPorT8/Hg²⁺ complex. The UV-vis absorption spectrum of a mixture of ZnPorT8 (2 μM) and Hg²⁺ (5.0 μM) in sodium cacodylate buffer (1.0 mM, pH 7.0) showed absorption bands at 428.6 nm (Soret band) and 558.8 nm (Q-band). The fluorescence spectrum of the ZnPorT8/Hg²⁺ system displayed two maxima at 604.0 nm and 654.0 nm when excited at 425.0 nm.¹⁸ Addition of L-Cys (5.0 μM) to the solution of ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) caused a blue shift of the Soret band from 428.6 nm to 425.0 nm together with small hyperchromicity (Fig. 2a, red curve). The fluorescence intensity of the ZnPorT8/Hg²⁺ complex at 654 nm (λ_exc = 425.0 nm) increased 6.21-fold from 130 a.u. to 810 a.u. upon the addition of L-Cys (5.0 μM; Fig. 2b, red curve). No cysteine induced shift of the emission maximum of ZnPorT8/Hg²⁺ complex was detected. Addition of L-Cys caused the release of the Hg²⁺ from its complex with ZnPorT8 and formation of a 1 : 1 L-Cys:Hg²⁺ complex.^48,49 Addition of D-Cys to ZnPorT8/Hg²⁺ system caused virtually identical spectroscopic changes as L-Cys and no chiral discrimination by ZnPorT8/Hg²⁺ system was observed (Fig. S6†). Interaction of GSH with ZnPorT8/Hg²⁺ complex promoted very similar spectroscopic changes as did Cys. Addition of GSH (5.0 μM) to the ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) caused a blue shift of the Soret band maximum from 428.6 nm to 424.8 nm accompanied by weak hyperchromicity in the UV-vis absorption spectrum (Fig. 2a, blue curve). The emission signal at 654 nm (λ_exc = 425.0 nm) increased without any observable λ_em shift 6.35-fold from 130 a.u. to 829 a.u. upon addition of GSH (5.0 μM; Fig. 2b, blue curve). Addition of GSH caused the dissociation of the ZnPorT8/Hg²⁺ and formation of a GSH:Hg²⁺ complex with 3 : 2 stoichiometry.⁴⁸ Addition of L-Cys (5.0 μM) or GSH (5.0 μM) to the solution of ZnPorT8 (2.0 μM) in the absence of Hg²⁺ did not cause detectable changes in the UV-vis absorption nor fluorescence spectra of the ZnPorT8 conjugate (Fig. S1 and S2†).

Fig. 2 (a) UV-vis absorption and (b) fluorescence spectra of the ZnPorT8 (2 μM, black dashed lines), ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM, black solid lines), ZnPorT8/Hg²⁺ in the presence of L-Cys (5.0 μM, red solid lines), and ZnPorT8/Hg²⁺ in the presence of GSH (5.0 μM, blue solid lines). Conditions: Na-cacodylate buffer (1.0 mM, pH = 7.0), λ_exc = 425 nm. Arrows depict the biothiol induced changes of the ZnPorT8/Hg²⁺ spectroscopic signal.

Absorption and emission spectral changes observed upon the addition of Cys or GSH to the ZnPorT8/Hg²⁺ complex are strong evidence of binding of biothiols to the Hg²⁺ ions of the ZnPorT8/Hg²⁺ complex via the thiol functional group.⁴⁸ Higher affinity of cysteine for Hg(II) than ZnPorT8 for Hg(II) caused the ZnPorT8/Hg²⁺ complex to fall apart thus releasing the strongly fluorescent ZnPorT8 conjugate. The comparison of spectroscopic characteristics of ZnPorT8/Hg²⁺ complex treated with biothiols (Fig. 2, red and blue curves) and ZnPorT8 conjugate (Fig. 2, black dashed lines) has provided final proof that additions of Cys and GSH released ZnPorT8 from its ZnPorT8/Hg²⁺ complex.

ZnPorT8/Cu²⁺ system: Cys and GSH optical sensing

The fluorescence of ZnPorT8 can also be quenched by Cu²⁺ ions, though less efficiently than with Hg²⁺. We have therefore explored the biomolecular detection of L-Cys and GSH by the ZnPorT8/Cu²⁺ supramolecular system. Fig. 3 shows the L-Cys-induced changes in absorption and fluorescence spectra of the ZnPorT8/Cu²⁺ system ([ZnPorT8] = 2.0 μM, [Cu²⁺] = 20.0 μM). Addition of L-Cys (6.0 μM) to the solution of ZnPorT8/Cu²⁺ complex ([ZnPorT8] = 2.0 μM, [Cu²⁺] = 20.0 μM) caused intense hypochromicity of the Soret band (65%), a red shift of the Soret band maximum from 425.8 nm to 428.6 nm (+2.8 nm; Fig. 3a, red curve) and appearance of a shoulder. Addition of Cys (6.0 μM; Fig. 3b, red curve) to ZnPorT8/Cu²⁺ complex induced fluorescence quenching as evidenced by the decrease of the emission intensity of the ZnPorT8/Cu²⁺ complex at 653 nm (λ_exc = 426.0 nm) by 87.7% from 653 a.u. to 80 a.u.

Fig. 3 (a) UV-vis absorption and (b) fluorescence spectra of the ZnPorT8/Cu²⁺ complex ([ZnPorT8] = 2.0 μM, [Cu²⁺] = 20.0 μM) in the absence of L-Cys (green curves) and in the presence of L-Cys ([L-Cys] = 6.0 μM; red curves). Conditions: Na-cacodylate buffer (1.0 mM, pH = 7.0), λ_exc = 426 nm. Arrows depict the biothiol induced changes of the ZnPorT8/Cu²⁺ spectroscopic signal.

Similar absorption and emission spectroscopic responses were observed upon addition of GSH (6 μM) to ZnPorT8/Cu²⁺ system (Fig. 3, blue dashed curves). Changes in absorption and emission spectra observed upon addition of Cys or GSH to the ZnPorT8/Cu²⁺ complex confirmed Cys and GSH interactions with Cu²⁺ in ZnPorT8/Cu²⁺ complex. Based on literature precedence,^19,50–52 we hypothesize that Cys and GSH partially reduced Cu(II) to Cu(I) with Cu(I) complex being a better quencher of ZnPorT8 emission than Cu(II).

It is important to emphasize that while the addition of L-Cys and GSH caused an enhancement of emission signal of the ZnPorT8/Hg²⁺ system, their addition to ZnPorT8/Cu²⁺ decreased the emission signal. The emission output (emission recovery or emission quenching) of the ZnPorT8/M²⁺ system could thus be easily reversed by complexed transition metal (mercury(II) vs. copper(II)) yielding tunable optical probe with excellent selectivity and sensitivity for Cys and GSH (Fig. 4). Importantly, absorption signal of the ZnPorT8/M²⁺ supramolecular system is also sensitive to the addition of biothiols. Addition of L-Cys and GSH to the ZnPorT8/Hg²⁺ system caused a hyperchromicity and a blue shift of the Soret band, their addition to ZnPorT8/Cu²⁺ gave rise to hypochromicity and a red shift. Both emission and absorption signals can thus be used for sensing of Cys and GSH resulting in a dual optical probe with switchable spectroscopic outcome.

Fig. 4 Schematic representation of transition metal induced switch of fluorescence response of ZnPorT8/Hg(II) and ZnPorT8/Cu(II) supramolecular systems to Cys and GSH.

ZnPorT8/Hg²⁺ and ZnPorT8/Cu²⁺: sensitivity of L-Cys and GSH detection using emission signals

To evaluate the sensitivity of the ZnPorT8/Hg²⁺ probe, we performed UV-vis absorption and fluorescence titration by adding L-Cys or GSH (1.0 μM to 10.0 μM) to the ZnPorT8/Hg²⁺ solution ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM). Fig. 5 shows the fluorescence titration of the L-Cys to ZnPorT8/Hg²⁺. The fluorescence intensity gradually increased with increasing concentration of the L-Cys and reached a plateau at [L-Cys] = 5.0 μM. We observed a linear correlation of fluorescence intensity at 654 nm as a function of the L-Cys concentration in the range between [L-Cys] = 0 and 5.0 μM with a slope of 136.79 (Fig. 5b). The emission limit of detection (LOD(em)) was determined to be [Cys] = 5.95 nM (3σ/slope, σ = 0.27). All detection limits are summarized in Table 1. Similarly to the L-Cys, fluorescence intensity of the ZnPorT8/Hg²⁺ system increased with the increasing concentration of the GSH with a plateau at [GSH] ≥ 3.0 μM (Fig. 5c). A linear correlation of 654 nm fluorescence signal as a function of the GSH concentration was observed between [GSH] = 0 and 3.0 μM (Fig. 5d). The LOD(em) for GSH was determined to be 3.34 nM (3σ/slope, σ = 0.27) with a slope of 243.47 (Fig. 5d).

Fig. 5 Fluorescence spectra (λ_exc = 425 nm) of the (ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) titrated with (a) L-Cys [L-Cys] = 1.0 μM to 10.0 μM in 1.0 μM addition steps) or (c) with GSH ([GSH] = 1.0 μM to 10.0 μM in 1.0 μM addition step). F − F₀ fluorescence intensity changes of the ZnPorT8/Hg²⁺ systems as a function of the (b) L-Cys or (d) GSH concentration (0 to 5.0 μM, black squares) detected at 654.0 nm and their linear fits (F − F₀, F₀: fluorescence intensity of ZnPorT8/Hg²⁺, F: fluorescence intensity ZnPorT8/Hg²⁺ after addition of biothiols). Arrows depict the biothiol induced changes of the ZnPorT8/Cu²⁺ spectroscopic signal.

Table 1 Emission and absorption detection limits of L-Cys and GSH

Amino acid^a	LOD	ZnPorT8/Hg^2+^a	ZnPorT8/Cu^2+^b
a [ZnPorT8] = 2.0 μM, [Hg^2+] = 5.0 μM; sodium cacodylate buffer (1 mM, pH 7.0).b [ZnPorT8] = 2.0 μM, [Cu^2+] = 20.0 μM; sodium cacodylate buffer (1 mM, pH 7.0).
L-Cys	LOD(em)	5.95 nM (enhancement)	11.99 nM (quenching)
	LOD(abs)	531 nM	263 nM
GSH	LOD(em)	3.34 nM (enhancement)	13.50 nM (quenching)
	LOD(abs)	511 nM	256 nM

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Stepwise addition of L-Cys (from 1.0 to 6.0 μM in 1.0 μM increments) to the ZnPorT8/Cu²⁺ system ([ZnPorT8] = 2.0 μM, [Cu²⁺] = 20.0 μM) caused a decrease of the fluorescence intensity at 654.0 nm by 79.9% (from 401 a.u. to 80 a.u.; Fig. 6a). A linear correlation of the 654.0 nm fluorescence signal as a function of concentration yielded LOD(em) for L-Cys of 11.99 nM (3σ/slope, σ = 0.3823, slope = 95.65, Fig. 6b). All detection limits are summarized in Table 1. Similarly, incremental addition of GSH (from 1.0 to 6.0 μM in 1.0 μM steps) induced quenching of fluorescence of ZnPorT8/Cu²⁺. The emission signal at 654 nm dropped by 80.3% (from 400 a.u. to 79 a.u.; Fig. 6c) yielding excellent sensitivity for the detection of GSH with LOD(em)_GSH = 13.50 nM (3σ/slope, σ = 0.3823, slope = 84.93, Fig. 6d).

Fig. 6 Fluorescence spectra (λ_exc = 425 nm) of the (ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Cu²⁺] = 20.0 μM) titrated with (a) L-Cys [L-Cys] = 1.0 μM to 6.0 μM in 1.0 μM addition steps) or (c) with GSH ([GSH] = 1.0 μM to 6.0 μM in 1.0 μM addition step). F − F₀ fluorescence intensity changes of the ZnPorT8/Cu²⁺ systems as a function of the (b) L-Cys or (d) GSH concentrations (0 to 3.0 μM, black squares) detected at 654.0 nm and their linear fits (F₀ − F, F₀: fluorescence intensity of ZnPorT8/Cu²⁺, F: fluorescence intensity ZnPorT8/Cu²⁺ after addition of biothiols). Arrows depict the biothiol induced changes of the ZnPorT8/Cu²⁺ spectroscopic signal.

ZnPorT8/Hg²⁺ and ZnPorT8/Cu²⁺: sensitivity of L-Cys and GSH detection using VIS absorption signals

Since the addition of Cys or GSH also caused significant changes in the UV-vis absorption spectra of ZnPorT8/Hg²⁺ and ZnPorT8/Cu²⁺ systems, we have explored the possibility to sense biological thiols by VIS absorption spectroscopy.

ZnPorT8/Hg²⁺ complex showed a blue shift of the Soret band from 428.6 nm to 425.0 nm with an isosbestic point at 427.0 nm upon stepwise addition of Cys (Fig. 7). A linear response of the absorption intensity at 425.0 nm as a function of the L-Cys ([L-Cys] = 0 and 5.0 μM) concentration was observed. The LOD(abs)_Cys was calculated to be 531 nM (3σ/slope, σ = 0.005, slope = 0.028, Fig. 7b). On the other hand, stepwise addition of Cys to ZnPorT8/Cu²⁺ system caused a red shift of the Soret band accompanied by hypochromicity (Fig. 7c). A linear response of the absorption intensity at 425.0 nm as a function of the L-Cys ([L-Cys] = 0 and 3.0 μM) yielded LOD(abs)_Cys of 531 nM (3σ/slope, σ = 0.005, slope = 0.028, Fig. 7d). The GSH LOD(abs) are summarized in Table 1. The absorption data confirmed that ZnPorT8/M²⁺ systems (M²⁺ = Hg²⁺ or Cu²⁺) can be used as dual optical probes for emission as well as absorption biomolecular recognition of biothiols. Lower emission LOD(em) in comparison to absorption LOD(abs) of ZnPorT8/M²⁺ systems originated from a larger change of emission signal that positively benefited from Cys- and GSH-induced change of absorbance signal at excitation wavelength (425.0 nm). All LODs are summarized in Table 1.

Fig. 7 UV-vis absorption spectra (Soret band) of the (a) ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) and (c) ZnPorT8/Cu²⁺ complex ([ZnPorT8] = 2.0 μM, [Cu²⁺] = 20.0 μM) titrated with L-Cys [L-Cys] = 1.0 μM to 10.0 μM in 1.0 μM addition steps. A − A₀ absorbance intensity changes of the (b) ZnPorT8/Hg²⁺ and (d) ZnPorT8/Cu²⁺ systems as a function of the L-Cys concentration (0 to 5.0 μM and 0 to 3.0 μM; black squares) detected at 425.0 nm and their linear fit (A − A₀, A₀: absorbance of ZnPorT8/Hg²⁺ or ZnPorT8/Cu²⁺, A: absorbance ZnPorT8/Hg²⁺ or ZnPorT8/Cu²⁺ after addition of L-Cys). Arrows depict the biothiol induced changes of the ZnPorT8/Cu²⁺ spectroscopic signal.

We have also examined the effect of concentration of Hg²⁺ in ZnPorT8/Hg²⁺ system on fluorescence cysteine detection limits (LOD(em)_Cys). Fig. S30† shows the fluorescence intensity changes detected at 654.0 nm of ZnPorT8/Hg²⁺ system ([ZnPorT8] = 2.0 μM) with five different concentrations of the Hg²⁺ ion (2.0, 3.0, 4.0, 5.0, and 6.0 μM) upon titration with Cys ([Cys] = 1.0 to 10 μM in 1.0 μM addition step). The LOD(em)_Cys have been determined to be 5.16 nM ([Hg²⁺] = 2.0 μM), 4.48 nM ([Hg²⁺] = 3.0 μM), 5.51 nM ([Hg²⁺] = 4.0 μM), 5.95 nM ([Hg²⁺] = 5.0 μM), and 6.39 nM ([Hg²⁺] = 6.0 μM) (Fig. S30†). The concentration of Hg²⁺ in ZnPorT8/Hg²⁺ system showed a clear potential to further tune the sensitivity of the supramolecular system towards biothiols.

ZnPorT8/Hg²⁺: selectivity of L-Cys and GSH detection

To evaluate the selectivity of ZnPorT8/Hg²⁺ complex for the spectroscopic detection of biothiols, the UV-vis absorption and emission spectroscopic changes were investigated with 17 other amino acids, i.e. Ala, Arg, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, and Trp. Fig. 8 illustrates the fluorescence response of ZnPorT8/Hg²⁺ complex at 654 nm (λ_exc = 425 nm) upon addition of different amino acids. UV-vis absorption and fluorescence spectra of the ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) showed very little changes upon the addition of any of these 17 amino acids, even at higher concentrations (up to 30 μM, Fig. S8–25†). Although the Hg²⁺ ion is known to bind to N-heterocycles, our data indicated that the Hg²⁺ affinity for ZnPorT8 is greater than for tested N-heterocyclic amino acids (i.e. Thr, His). ZnPorT8/Hg²⁺ system exhibited excellent sensitivity for L-Cys and GSH over other amino acids.

Fig. 8 Fluorescence intensity changes of solution of the ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) at 654.0 nm upon addition of amino acids ([Cys] = 5.0 μM, [GSH] = 5.0 μM, [other amino acids] = 10.0 μM).

Next, we evaluated the performance of the ZnPorT8/Hg²⁺ system under conditions in which several analytes were present at high concentrations (competition experiments). We prepared a mixture of amino acids containing Ala, Lys, Met, Pro and Trp, and we also made another mixture of the same amino acids that also contained L-Cys. The concentration of each amino acid was set to 5.0 μM (total amino acid concentrations were 30.0 μM and 25.0 μM, respectively). We added aliquots of these amino acid mixtures to the ZnPorT8/Hg²⁺ complex ([ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM) and recorded absorption and fluorescence spectroscopic changes (ESI, Fig. S31†). Addition of the amino acid mixture without L-Cys did not show any evident spectroscopic changes. On the other hand, the mixture containing L-Cys caused very similar spectroscopic changes to neat L-Cys: the blue shift of the Soret absorption band from 428.6 to 425.0 nm and an increase of the fluorescence signal recorded at 654 nm (λ_exc = 425.0 nm). The results of the competition experiments confirmed that non-thiol amino acids did not interfere with L-Cys sensing by using the absorption and emission spectroscopic responses of the ZnPorT8/Hg²⁺ complex.

Experimental

Materials

Amino acids (L-Cys, D-Cys, L-GSH, Ala, Arg, Asn, Gln, Gly, Glu, His, Ile, Leu, Lys, Met, Pro, Phe, Ser, Thr, Tyr, and Trp), Hg(ClO₄)₂, Cu(ClO₄)₂, and sodium cacodylate were purchased from Sigma-Aldrich. Ultrapure water was obtained from a Milli-Q system with a resistivity of 18.2 MΩ cm. Synthesis, purification and characterization of zinc(II)porphyrin–oligothymine conjugate ZnPorT8 has been reported previously.^53,54

Methods

The stock solutions of ZnPorT8, Hg²⁺, Cu²⁺, and all amino acids were prepared in a sodium cacodylate buffer (1 mM, pH 7.0). The concentration of ZnPorT8 stock solution was determined by UV-vis absorption spectroscopy using the extinction coefficient ε_ZnPorT8 = 2.9 × 10⁵ M⁻¹ cm⁻¹ at 425 nm.^53,54 The fluorescence and absorption titration experiments were performed as follows: aliquots of amino acids (0.8 μL, concentration of the stock solutions = 1 mM) were added into a 800 μL solution of the ZnPorT8/M²⁺ complex (M²⁺ = Hg²⁺ or Cu²⁺, [ZnPorT8] = 2.0 μM, [Hg²⁺] = 5.0 μM, [Cu²⁺] = 20.0 μM) in the sodium cacodylate buffer (1 mM, pH = 7.0). The absorption and emission spectra were recorded upon each addition. The collected spectroscopic data have been corrected for dilution (in all cases dilution was kept below 1%).

Instrumentation

UV-vis absorption spectra were collected at 20 °C using a Jasco V-650 UV-vis double beam spectrophotometer equipped with a single position Peltier temperature control system. Fluorescence measurements were performed at 20 °C on a Varian Cary Eclipse fluorescence spectrophotometer equipped with a Peltier temperature control system. The emission spectra were collected from 500 to 800 nm with an excitation wavelength of 425.0 nm. Conditions were as follows: excitation slit 10 nm, emission slit 10 nm, scan rate 600 nm min⁻¹. A quartz cuvette with a 1 cm path length was used for all absorption and emission experiments.

Conclusions

We have reported a tunable supramolecular system assembled from the zinc(II)porphyrin–oligothymidine conjugate (ZnPorT8) and a transition metal ion as a dual (emission/absorption) highly selective and sensitive optical probe for biothiol. We have shown that the fluorescence as well as absorption response of ZnPorT8 can be switched by a complexed metal ion (either Hg(II) or Cu(II)). The ZnPorT8/Hg²⁺ complex offered a turn-on fluorescence sensing of Cys and GSH (fluorescence enhancement), while the ZnPorT8/Cu²⁺ system exhibited reversed turn-off sensing (fluorescence quenching). Both turn-on as well as turn-off biomolecular optical detection can be achieved using the same bioorganic scaffold. Importantly, while the transition metal ions modulated the optical outcome, they had only marginal effect on the sensitivity of the ZnPorT8/M²⁺ supramolecular system. Both complexes (ZnPorT8/Hg²⁺ and ZnPorT8/Cu²⁺) displayed excellent limit of detection using emission signal for Cys (LOD(em) = 5.95 nM and 11.99 nM, respectively) and GSH (LOD(em) = 3.34 nM and 13.50 nM, respectively) using the 3σ/slope method. The other 17 proteinogenic amino acids did not cause any significant fluorescence or absorption signal changes when added to ZnPorT8/Hg²⁺.

Acknowledgements

We thank UW SER Graduate Assistantship (GS, MB), Center for Photoconversion and Catalysis (MB), Department of Chemistry Graduate Assistantship (JKC) and Wyoming INBRE undergraduate research fellowship (BDJ). We thank Prof. Ed Clennan (Department of Chemistry, UW) for acquisition of and access to the Varian Cary Eclipse fluorescence spectrophotometer.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: UV-vis absorption and emission spectra of ZnPorT8/Hg²⁺ and ZnPorT8/Cu²⁺ complexes titrated with different amino acids. See DOI: 10.1039/c4ra16453a

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