Colorimetric and Fluorescent Sensing of SCN- Based on meso-Tetraphenylporphyrin/meso-Tetraphenylporphyrin Cobalt(II) System.

An approach for colorimetric and fluorescent sensing of thiocyanate (SCN-) has been proposed based on the competitive-displacement strategy between meso-tetraphenylporphyrin (TPP) and meso-tetraphenylporphyrin cobalt(II) (CoTPP). In THF-water solution, TPP emits strong fluorescence at 651 nm; however, the fluorescence was quenched stepwise by CoTPP, and then restored by SCN-, the detection limit is 6.0 × 10-4 M. The recognition of SCN- could also be easily achieved by visual way since the assembly system showed significant color change by the anion. Both the fluorescence and the color change of the system exhibits remarkably high selectivity to SCN- over a large series of anions. The interaction mechanisms among TPP, CoTPP and SCN- were primarily investigated by fluorescence lifetime. The quenching of TPP fluorescence is attributed to the formation of TPP/CoTPP aggregates, and the fluorescence restoration is due to the binding of CoTPP with SCN-, releasing the free TPP. This simple system has the potential to be used as a latent fluorescent sensing approach for SCN- for environmental analysis.


Introduction
There has been growing interests in the design of synthetic receptors for anions because of the important role of the analytes in biological and environmental fields, as well as food science [1]. As one of the important inorganic anions, SCNhas made a great influence on both environment and the human body. It has been widely used in fabric dyeing, photographing, electroplating baths and so on [2]. SCNis also known to block the iodine uptake by the thyroid gland [3,4]. Several methods, such as chromatography [4,5], amperometry [3] and ion-selective electrodes [6,7], etc. have been previously reported in the literature for the assay of SCN -. However, most of them require either sophisticated operation or time-consuming synthetic efforts. Therefore, there is still need for the development of a simple method for multipurpose determination of SCN -.
Fluorescent sensor has its own advantages such as high sensitivity and easy operation [8]. In general, a fluorescent sensory molecule would involve the covalent linking of a receptor domain to a fluorescent fragment (i.e. the signaling unit) [9][10][11][12]. However, this synthetic work required elaborate design of the synthetic routes and many heavy and complicated synthetic steps. In this respect, the development of straightforward, low-cost and simple system for particular analytes should be of general interests. One is the use of non-covalent assembly approaches based on the competition indicator displacement between a fluorophore bonded to a receptor and a certain analyte [13][14][15]. When the receptor and fluorophore are carefully chosen, selective sensing event can be achieved.
Metalloporphyrins are one of the attractive candidates for anions receptor due to the high binding affinity of the metal center with the analytes [16][17][18][19]. It was found that in organic medium, the TPP fluorescence could be quenched in the presence of some metalloporphyrins [20,21]. However, it has been taken little attention to restore the quenched fluorescence by other substrates which may be a potential anions sensing approach. With this in mind, our goal is to investigate whether this quenched TPP fluorescence can be restored, and further be used as a fluorescence recognition approach for anions in a suitable way. In this contribution, we reported our study on the interaction of TPP and CoTPP to develop an efficient colorimetric and fluorescent enhancement sensor for SCN -. In the present case, CoTPP was used as SCNreceptor and TPP acted as a fluorescent reporting unit, which does not require establishing any covalent linking between the fluorophore and the receptor but utilizes the fluorophore and receptor as such.

Fluorescence Quenching of TPP by Metalloporphyrins
Prior to application in fluorescence sensing of SCNanions, the effects of metalloporphyrins including meso-tetraphenylporphyrin zinc(II) (ZnTPP), meso-tetraphenylporphyrin manganese(II) (MnTPP) and CoTPP on the TPP fluorescence were first studied in the THF-water solution ( Figure 1). In the absence of metalloporphyrin, TPP exhibited a strong emission peak at 651 nm. When CoTPP or MnTPP was added into the TPP solution, the TPP fluorescence significantly decreased. On the other hand, upon the addition of ZnTPP to the TPP solution, the fluorescence at 651 nm decreased concomitant with a new emission centered around 600 nm appearing, which might contribute to the emission of ZnTPP [22]. The quenching efficiencies, Ksv, of TPP by the three metalloporphyrins are 4.01 × 10 4 M -1 (MnTPP), 1.54 × 10 5 (ZnTPP), and 3.14 × 10 5 (CoTPP). CoTPP display the strongest quenching.

Restoration of TPP/CoTPP by Anions
To explore the utility of the TPP/CoTPP as anion-sensing approaches, the fluorescence responses of the system to anions were tested. It was found that the quenched fluorescence of TPP could be restored by anions.

Binding Iteractions of CoTPP with Anions and SCN -Selectivity
To further clear out the high selective fluorescence response of TPP/CoTPP to SCN -, the binding interactions of CoTPP with anions were studied by UV-Vis spectroscopy. In the THF-water (8/2, v/v) solution, the potassium salts of Cl -, CO   The UV-Vis spectroscopy of CoTPP was followed as aliquots of SCNwere added to the THF aqueous solutions of CoTPP (Figure 4). Stepwise addition of SCNled to significant decrease in the CoTPP absorption band at 416 nm with the concomitant formation of a new red-shifted absorption band at 442 nm through a clear isosbestic point at 426 nm. The well-defined isosbestic point indicates that a neat interconversion between the uncomplexed and complexed species occurs. By following the absorption change at 442 nm and a curve fitting analysis [23][24], the stoichiometry of CoTPP with SCNwas estimated to be 1: 2 (n = 2, which denotes the association ratio of SCNto CoTPP), the corresponding association constant K, is 9.85 × 10 3 mol -2 L 2 (Figure 4, inset). The association constants of CoTPP with other anions are summarized in Table 1, CoTPP shows strongest affinity with SCNover other anions, which is the result of highly selective fluorescence response of TPP/CoTPP to SCN -.   Interestingly, the solution was initially pale red in color and changed to deep yellow when exposed to SCN -( Figure 5). The color would go deeply as time passed. NO 2 showed very pale green color which is consistent with the very slim change on UV-Vis spectrum. The results indicate that color changes can be used for a "naked-eye" detection of SCN -.

Mechanism Studies for the Fluorescence Quenching and Restoration of TPP
In our experiment, it was not found that Zn 2+ , Co 2+ or Mn 2+ significantly quenched the TPP fluorescence, which demonstrated that the fluorescence quenching of TPP by the metalloporphyrins was related to the interactions of the two porphyrins rather than the heavy atom effects. Fluorescence lifetime measurements were carried out to understand the mechanism of TPP fluorescence quenching and restoration. From Table 2 it can be seen that in the presence of CoTPP, the fluorescence lifetime of TPP is almost unaffected, which suggests that the quenching between TPP and CoTPP contributes to the formation of TPP/CoTPP ground state aggregation instead of electron transfer (PET) or excited energy transfer. Because if it is PET process, electrons of the free-base porphyrin in singlet excited state will transfer to a low-spin empty orbit of cobalt(II) [25], which means the presence of CoTPP will undoubtedly decrease the fluorescence lifetime of TPP. In contrast, upon addition of SCN -, it shows a slim lifetime enhancement. This result may due to the stabilization of the TPP excited state caused by SCN -. In all the measurements the best fits were obtained by monoexponential decay functions, which indicated the homogeneity of the chromophore in the excited state [26].

Preliminary Application
At the optimum experimental condition, the preliminary application of the present approach was tested by applying it in the determination of SCNconcentration in three synthetic samples in the presence of other interferents. The synthetic samples were prepared on the basis of possible existing anions in physiological environment. The fluorescence titrations were carried out by adding a few microliters of the synthetic samples to TPP/CoTPP system, and the results were compared with those of SCNsolutions without the interferents. The results are given in Table 3, and the recoveries of the three samples are 98.8~107.4 %.

Conclusion
In conclusion, a simple and efficient sensing approach capable of high selective colorimetric and fluorescent sensing of SCNhas been proposed. In contrast to the traditional design of molecular receptors as chemical sensors based on geometric and electronic complementarity of the fluorophore-appended receptor to match that of the analyte, this simple approach needs no tedious synthetic efforts and obtains high selectivity. It has the potential to be explored as a SCNcolorimetric and fluorescent sensing system. Furthermore, from a mechanism perspective, it will be helpful for providing more information to better understanding of porphyrin-based chemistry as well as, or at least the interactions between porphyrin units and anions.

Materials
TPP and all kinds of metalloporphyrins were synthesized and purified in our laboratory according to the literatures [27][28][29]. The 1.0 × 10 -4 mol L -1 stock solutions of porphyrins were prepared in THF. The concentrations of the porphyrins were determined by spectrophotometrically by using Beer's law and the corresponding absorptivity values. Below 4.0 × 10 -6 mol L -1 , the TPP absorption values in the THF-water (8/2, v/v) solution was increased with increasing in the TPP concentration, indicating there were no aggregations of TPP [30]. All organic solvents and the potassium salts of all kinds of anions were of analytical-grade. All salt solutions were prepared by doubly distilled water. The working solutions of the porphyrins were obtained by diluting the stock solution with THF and doubly distilled water. The substances described above were used as available without further purification.

Methods
Fluorescence emission spectra were acquired on a Hitachi F-4500 fluorescence spectrofluorometer (Kyoto, Japan) with the excitation and emission slits of 10.0 nm. Fluorescence lifetime measurements were carried out on Edinburgh Instruments FLS 920 (UK). The concentrations of TPP and CoTPP were 2.5 × 10 -6 mol L -1 and 5.0 × 10 -6 mol L -1 , respectively. The excitation and emission wavelength was 416 and 651 nm, respectively. UV-Vis spectra were taken on a Hitachi U-3010 UV-Vis spectrophotometer (Kyoto, Japan). The fluorescence and absorption titrations of TPP/CoTPP system with the analytes were carried out by adding a few microliters of a stock solution of the materials to 1.0 mL of TPP/CoTPP solution with a quartz cell (1.0 × 1.0 cm 2 cross section). 1 HNMR spectra were recorded on an Invoa-400 spectrometer at 298 K Samples for 1 H NMR analysis were prepared in 5 mm NMR tubes using CDCl 3 and CDCl 3 /CD 3 COCD 3 as solvent in the absence and presence of KSCN with tetramethylsilane (TMS) as internal standard.