A ratiometric fluorescence probe for the selective detection of H2S in serum using a pyrene-DPA–Cd2+ complex

A ratiometric and selective hydrogen sulfide (H2S) detection probe was proposed based on the pyrene-DPA–Cd2+ complex through the metal ion displacement approach (MDA) mechanism. While most MDA-based fluorescence probes with paramagnetic Cu2+ have focused on the development of a simple turn-on sensor using the broad spectral range of fluorescence enhancement, this ratiometric probe exhibited unchanged monomer emission as a built-in internal reference with an increase in excimer emission with added H2S. The demonstrated probe showed a rapid response (within 1 min) and a high sensitivity, with 70 nM as the limit of detection. The selectivity for H2S over cysteine, homocysteine and glutathione was confirmed, and reliable fluorescence enhancement, which could be monitored by the naked eye, was observed upon irradiation with handheld UV light. In addition, this detection system was successfully applied to detect H2S in human serum without interference from biological molecules.


Introduction
Hydrogen sulde (H 2 S) is a gaseous signaling molecule that has received attention because of its toxicity for physiological functions in humans. 1,2 Endogenous H 2 S, which is generated mainly from cysteine (Cys), homocysteine (Hcy), and 3-mercaptopyruvate by enzymatic reactions of cystathionine b-synthase, cystathioine-g-lyase, and 3-mercaptopyruvate sufurtransferase/cysteine aminotransferase, is involved in various biological processes such as blood pressure regulation, metabolic disorders, neurodegeneration, and inammation. [3][4][5][6][7][8] Therefore, the imbalanced synthesis of endogenous H 2 S is related to some human diseases such as lung infection, Alzheimer's disease, and diabetes. [9][10][11] Thus, abnormal levels of endogenous H 2 S in human serum can be used as evidence for such diseases. Considering the importance of H 2 S as a biomarker, various detection methods have been developed for the accurate measurement of changed endogenous H 2 S based on chromatography, electrochemical techniques, colorimetric, and uorescent chemosensors. [12][13][14][15] Among them, uorescence detection methods have shown favorable properties such as rapid response, easy sample preparation, and high sensitivity, and various examples have been reported recently.
The currently known methods for uorescent H 2 S detection were designed by using the chemical properties of H 2 S. 16,17 These detection methods typically employ four types of probes depending on the recognition mechanism used by them; they may be based on nucleophilicity, reduction, metal coordination, or the metal displacement approach (MDA). [18][19][20][21] MDA is a strategy that exploits uorescence changes caused by the removal of metal ions, which coordinate to a uorophore through metal ions and H 2 S complex formation. Owing to the coordination with charged metal ions and the high binding affinity between metal ions and H 2 S, MDA has several advantages over other mechanisms, including good water solubility, rapid response time, and high sensitivity. 22 Therefore, MDA is a detection mechanism that is appropriate for application in various environments. Although some metal ions, including Cd 2+ , Zn 2+ , Hg 2+ , and Cu 2+ , show very low solubility products (K sp ) when bound to H 2 S, most proposed examples have focused on paramagnetic Cu 2+ because uorescence recovery through CuS complex formation promotes turn-on uorescence detection aer uorophores are quenched by Cu 2+ binding; this turnon detection method displays fewer false positive signals and good distinguishability from the background compared to the turn-off method. 23,24 Consequently, examples of MDA-based H 2 S detection using other metal ions have been limited to date.
Ratiometric uorescence probes emit two or more at different wavelengths, and the ratio between the emissions is used to detect the target molecules. Ratiometric probes have unique features that enable self-calibration for the quantication of the target, and they also have built-in correction systems that reduce the environmental interference and enable them to have high signal-to-noise ratios. 25,26 Therefore, numerous applications of these probes for various analytes have been developed, including H 2 S detection. 27,28 However, despite the advantages that make MDA among the four typical H 2 S detection mechanisms, this mechanism has not garnered attention as a strategy for the development of ratiometric detection methods except for a little example. 29,30 This is because quenching the entire spectral range of uorescence by paramagnetic Cu 2+ induces only simple uorescence enhancement instead of ratiometric changes upon addition of H 2 S. In addition, the excessively strong interaction between thiol and Cu 2+ causes other biothiols such as Cys, Hcy, and glutathione (GSH) to interfere with the reactions and reduce the selectivity of H 2 S detection, as shown in several previous studies. 22 Thus, it is crucial to develop ratiometric uorescent probes for the detection of H 2 S using other metal ions that retain the advantages of the MDA-based mechanism and also display great selectivity of H 2 S over other biothiols.
In this study, the pyrene-DPA-Cd 2+ complex was proposed for use in a ratiometric uorescence probe for H 2 S detection that is selective to other biothiols (Scheme 1). Previous studies performed in our group revealed that coordination of pyrene-DPA with Zn 2+ decreased excimer emission without changing the monomer emission. 31 Inspired by this result, it is expected that a complex of pyrene-DPA with a metal ion that has high binding affinity to H 2 S and quenches an excimer emission can be applied to develop ratiometric detection methods based on the MDA using the unchanged monomer as a built-in internal standard. Screening the response of the pyrene-DPA-metal ion complex in the presence of various biothiols, it was conrmed that Cd 2+ , which is in the same group as Zn 2+ , reduced only the excimer emission of pyrene-DPA; in particular, other biothiols apart from H 2 S did not inuence any emissions in the pyrene-DPA-Cd 2+ complex. Thus, the pyrene-DPA-Cd 2+ complex could be used as a ratiometric probe for selective H 2 S detection over biothiols with high sensitivity and applicability in real serum samples, which were derived from the features of the MDA mechanism.

Materials and instrumentations
Chemical reagents were purchased from commercial sources (Sigma Aldrich, Alfa Aesar, Duksan, and Tokyo Chemical Industry) and used without further purication. Human serum was purchased from Sigma Aldrich (catalog number: H6914). 1 H NMR spectra were recorded on a JEOL 400 MHz NMR spectrometer. Fluorescence spectra were measured on Agilent Cary Eclipse uorescence spectrometer.

Synthesis of pyrene-DPA and in situ generation of pyrene-DPA-metal ion complex
The pyrene-DPA compound was prepared from previous literature of our group. 31 For in situ pyrene-DPA-Cd 2+ complex, same volume of pyrene-DPA and metal ion solution with same concentration were added to the buffer solution for each measurements and incubated for 5 min. Aer the incubation, various analytes were introduced and uorescence or NMR spectra were obtained.
For selection of optimal pH condition, H 2 S and Cys (60 mM) were added to buffer solution (20 mM, pH 5.0 acetate, pH 6.0 MES, pH 7.0, 7.4, and 8.0 HEPES, pH 9.0 Tris) containing pyrene-DPA-Cd 2+ complex (20 mM) and uorescence spectra was recorded with excitation at 341 nm. containing pyrene-DPA-Cd 2+ complex (20 mM) and uorescence spectra was obtained with excitation at 341 nm. Limit of detection (LOD) was estimated by 3s/S using excimer enhancement against monomer emission from 0 to 1.0 mM H 2 S where s is standard deviation of blank and S means slope from titration data for H 2 S detection.

Results and discussion
Screening for selection of an optimal metal ion and pH conditions To develop a ratiometric probe for the selective detection of H 2 S over other biothiols, uorescence responses through metal ion displacement of pyrene-DPA complexes were compared for various metal ions along with the addition of biothiols. In the case of Cu 2+ and Hg 2+ , the entire uorescence-containing monomer and excimer emission were reduced during complex formation between metal ions with pyrene-DPA (Fig. S1 †). Therefore, they were not suitable for the development of ratiometric detection probes. Dramatic enhancement of excimer emission at 476 nm against monomer emission at 376 nm (F 476 /F 376 ) was observed only in the pyrene-DPA-Zn 2+ or Cd 2+ complex with selectivity for H 2 S over Cys, Hcy, and GSH when excess of biothiols (5 equiv.) was added to the pyrene-DPA-metal ion complex (Fig. 1). Considering that the pyrene-DPA-Zn 2+ complex was previously used as a probe for the detection of PPi, which is abundant in a biological environment, uorescence changes under interference from PPi were measured (Fig. S2 †). 32 Unlike the pyrene-DPA-Cd 2+ complex, the pyrene-DPA-Zn 2+ complex displayed an increase in excimer emission in the presence of PPi. This property of the pyrene-DPA-Zn 2+ complex would hinder its application in the detection of H 2 S in real samples. Therefore, the pyrene-DPA-Cd 2+ complex was chosen as an optimal probe for selective and ratiometric H 2 S detection. The optimal pH conditions were determined by evaluating the uorescence responses of the pyrene-DPA-Cd 2+ complex for H 2 S and Cys because in human serum, the approximate Cys concentration is higher than that of H 2 S, which makes it difficult to distinguish them (Fig. S3 †). 33 At pH values above 7.4, interference of Cys was observed, and this interference increased with the pH. A more drastic change in uorescence induced by Cys was observed at pH 9.0. At neutral pH, the uorescence enhancement by H 2 S was still similar to that at basic pH, and interference by Cys was inexistent. As a result, a pH of 7.0 was used in subsequent experiments.

Sensitivity for detection of H 2 S
The uorescence change of the pyrene-DPA-Cd 2+ complex with various concentrations of H 2 S was monitored to verify the sensitivity and the available detection range of the developed probe. The excimer emission at 476 nm upon addition of H 2 S with unchanged monomer emission at 376 nm increased gradually with increasing the H 2 S concentrations (Fig. 2a). The extent of uorescence change in the excimer against monomer emission as an internal reference exhibited an almost linear relationship with the concentration of H 2 S (Fig. 2b). The largest enhancement was observed with 20 mM of H 2 S because the upper limit of detection was dependent on the concentration (20 mM) of pyrene-DPA-Cd 2+ complex (Fig. 2b). As expected, the MDA-based mechanism enables rapid detection and high sensitivity; the uorescence change was observed within 1 min, and LOD was estimated to be 70 nM from titration data ( Fig. S4 and S5 †).

Selectivity and interference test for detection of H 2 S
In a biological environment, biothiols and various anions can disturb the detection of H 2 S. To investigate the selectivity of the suggested probe, spectral changes of the pyrene-DPA-Cd 2+ complex in the presence of these compounds were examined. The biothiols, except for H 2 S, did not affect the uorescence intensity of the pyrene-DPA-Cd 2+ complex, and the entire uorescence spectra of the complex did not change upon the addition of various anions (Fig. 3a). In particular, the uorescence responses with biothiols were photographed under irradiation with handheld UV light, and only H 2 S showed a reliable uorescence enhancement that can be observed by the naked eye (Fig. 3a). To explore the interference for the probe from biothiols and anions, the detection of H 2 S in the presence of excess biothiols and various anions (3 equiv.) was performed. The coexistence of other biothiols or most anions did not induce a notable interference in the detection of H 2 S, while a slight additional increase in excimer emission with PPi was observed (Fig. 3b). Thus, it is expected that this great selectivity and unimpeded features to detect H 2 S would allow its application in a real serum sample.

Mechanistic study for H 2 S detection based on metal ion displacement
To evaluate the mechanism for the detection of H 2 S based on the pyrene-DPA-Cd 2+ complex, 1 H-NMR spectra along with Cd 2+ and subsequent H 2 S addition to pyrene-DPA were compared (Fig. 4). The assignment of the proton signal in pyrene-DPA was proposed in a previous report. 34 The aromatic proton signal (H a , H b , H c , and H d ) in the DPA group, which is a well-known metal ion binding moiety, was shied downeld following the addition of Cd 2+ , suggesting that the strong electron-withdrawing Cd 2+ was coordinated to the DPA group and reduced the electron shielding effect. In succession, the introduction of H 2 S in the pyrene-DPA-Cd 2+ complex facilitated the recovery of the downeld protons to the initial signal similar to that of pyrene-DPA, regarded as a displacement of Cd 2+ through the formation of a CdS complex. 35 In addition, the aliphatic proton (H e ) near the pyrene unit was moved downeld while coordinating with Cd 2+ and recovered again with subsequent H 2 S addition, indicating that effective p-p stacking between pyrene was removed by Cd 2+ addition and recovered by H 2 S addition. 36 Thus, it could be inferred that the uorescence excimer emission recovery aer quenching on the coordination of Cd 2+ was caused by the change in distance between pyrene groups due to Cd 2+ displacement with H 2 S through the MDA mechanism.

Application of H 2 S detection in human serum samples
The utility of the developed system in a practical sample was conrmed using spiked H 2 S human serum samples. Human serum samples with various concentrations of spiked H 2 S were prepared through a pretreatment process to remove proteins. The uorescence change caused by the spiked H 2 S was used to estimate the concentrations, and the results were compared with the known values. As summarized in Table 1, the obtained H 2 S concentration from the probe showed recoveries ranging  from 94.8% to 110.1%. Moreover, H 2 S detection by the demonstrated detection system was unaffected by most molecules present in human serum, and these results show its potential for accurately and precisely detecting H 2 S in human serum.

Conclusions
We developed a ratiometric uorescence probe for selective H 2 S detection based on the pyrene-DPA-Cd 2+ complex, which works based on the MDA mechanism. The increase in excimer emission against a xed monomer emission, which was used an internal reference, facilitated ratiometric detection of H 2 S, which was previously difficult using the MDA strategy. Using the MDA, rapid response (within 1 min) and high sensitivity (LOD ¼ 70 nM) for the detection of analytes were conrmed. The selectivity for H 2 S over Cys, Hcy, and GSH was exhibited without interference from these molecules, and the selectivity over other biothiols was evaluated through the signicant uorescence enhancement monitored by the naked eye upon irradiation with handheld UV light. Moreover, H 2 S detection in human serum was achieved without interference from other components. Thus, the demonstrated system shows potential for use in practical applications.

Conflicts of interest
There are no conicts to declare.