A colorimetric and fluorescent signaling probe for assaying Pd2+ in practical samples

We developed an optical signaling probe to detect Pd2+ ions in Pd-containing catalyst and drug candidate. The Pd2+ signaling probe (Res-DT) was readily prepared by reacting the versatile fluorochrome resorufin with phenyl chlorodithioformate. In a phosphate-buffered saline solution (pH 7.4) containing sodium dodecyl sulfate (SDS) as a signal-boosting surfactant, Res-DT exhibited a pronounced colorimetric response with a chromogenic yellow to magenta shift, leading to a substantial increase in the fluorescence intensity. The Pd2+ signaling performance of Res-DT was attributed to the Pd2+-promoted hydrolysis of the dithioate moiety. The probe displayed high selectivity toward Pd2+ ions and remained unaffected by commonly encountered coexisting components. Moreover, the detection limit of Res-DT for Pd2+ ions was 10 nM, and the signaling was achieved within 7 min. Furthermore, to demonstrate the real-world applicability of Res-DT, a Pd2+ assay was performed in Pd-containing catalyst and drug candidate using an office scanner as an easily accessible measurement device. Our results highlight the prospects of Res-DT as a tool to detect Pd2+ ions in various practical samples, with potential applications in catalysis, medicine, and environmental science.


Introduction
Palladium (Pd) is a versatile and valuable Pt-group metal with diverse practical applications in industrial arenas such as dentistry, electronics, chemical synthesis, groundwater treatment, and exhaust gas treatment. 1,2Additionally, it is employed in therapeutics owing to its antiviral, antifungal, antimicrobial, anticancer, and cardioprotective properties. 3Furthermore, it is a vital component of fuel cells that generate energy through chemical reactions involving hydrogen and oxygen. 4d-based catalysts are essential for producing myriad ne chemicals that are used to manufacture pharmaceuticals and agricultural products. 5In particular, several Pd-catalyzed organic reactions, including the Mizoroki-Heck reaction, 6 Suzuki-Miyaura reaction, 7 Sonogashira-Hagihara reaction, 8 Buchwald-Hartwig amination, 9 carbonylation, 10 and cyanation, 11 are performed in pharmaceutical preparations. 12Moreover, Pd complexes can be used as anticancer agents owing to their similar chemical and physical properties to those of the widely employed Pt complexes. 13However, the inherent toxicity of Pd has made Pd contamination a matter of considerable concern. 14In particular, Pd species can bind to important biological materials, such as amino acids, DNA, RNA, and proteins, 15,16 thereby disrupting cellular processes and leading to severe health problems including weight loss, muscle weakness, seizures, and heart disease. 17Consequently, the determination of residual Pd in commercially available drug chemicals, agricultural products, and foods is crucial.
Various conventional analytical techniques have been employed to detect Pd in different analytes using sophisticated, specialized analytical instruments. 18However, these techniques typically require complex sample preparation protocols, stringent experimental conditions, and highly skilled operators. 19In contrast, colorimetric or uorescent chemosensors and reaction-based probes hold greater promise for selective and sensitive metal ion/anion detection, including Pd.These methods offer straightforward operability, high sensitivity, no need for heavy instruments, and widespread applicability. 20o meet the increasing need for simpler and more convenient Pd analysis methods, several colorimetry or uorescencebased chemosensors and reaction-based probes have been developed. 19,21Pd signaling sensors have been obtained using diverse ligands including pyridine-2,6-dicarboxamide, 22 2chloroethyl methyl sulde, 23 purine derivative, 24 and 2-picolylamine. 25Additionally, several Pd-selective signaling probes have been developed by leveraging their Pd-selective reactions, signal-accumulating ability, and ease of design. 26For instance, deallylation of allyl carbamates, 27 allyl carbonates, 28 allyl ethers, 29 and allyl ester, 30 and depropargylation of propargyl ethers 31 and propargyl carbamates 32 have been extensively performed to design Pd signaling probes.In addition, probes that detect Pd via metal-induced organic transformations have been prepared using the Claisen rearrangement, 33 dimerization, 34 and oxidative cyclization reactions. 35Furthermore, several Pd signaling probes that exploit the hydrolysis of thiocarbamate and hydrazones of resorun and rhodamine uorochromes have been engineered. 36There have also been various studies exploring Pd 2+ sensing probes, each contributing valuable insights into their development and applications. 37Representative optical Pd specic reaction-based probes have been summarized in Table S1 (ESI).† In this study, we introduce a novel dual-mode probe (Res-DT) designed for the highly sensitive detection of Pd 2+ ions through both colorimetric and uorescent responses, facilitated by the hydrolysis of dithioate-modied resorun.The probe ensures rapid, convenient, and naked-eye detectable responses, obviating the need for complex instrumentation.Res-DT demonstrates efficiency in swily and precisely assaying residual Pd 2+ in a Pd-containing catalyst and a Pd-containing drug candidate noted for its antimicrobial and anticancer activities.

Synthesis of Res-DT
Resorun dithioate (Res-DT) was synthesized using a previously reported method with slight modications. 38In a 100 mL round bottom ask, resorun (0.43 g, 2.0 mmol) was dissolved in 30 mL of N,N-dimethylformamide (DMF).The solution was then mixed with triethylamine (TEA; 0.56 mL, 4.0 mmol) and stirred for 30 min at room temperature.Phenyl chlorodithioformate (0.45 mL, 3.0 mmol) was then added carefully to the solution, and the reaction was allowed to continue for 12 h with constant stirring.DMF was then removed by passing air over the system, and the remaining solid was dissolved in dichloromethane (50 mL).The resulting solution was washed with distilled water and brine and then evaporated.The obtained residue was puried by column chromatography (CH 2 Cl 2 : CH 3 OH = 49 : 1, v/v).Res-DT.0.52 g, 71% yield as a vermillion-colored powder. 1

Stock solution preparation
A stock solution containing probe Res-DT (0.5 mM) was made by dissolving Res-DT in dimethyl sulfoxide (DMSO).Pdcontaining solutions (5.0 mM) were prepared by dissolving Pd(OAc) 2 , PdCl 2 , and Pd(PPh 3 ) 4 in DMSO, and K 2 PdCl 6 in deionized (DI) water.Solutions of metal ions and anions (5.0 mM) were prepared using metal perchlorate salts and sodium salts of the anions, respectively, in DI water.Oxidant solutions (5.0 mM), including H 2 O 2 , HOCl, peracetic acid, O 2 − , perborate, percarbonate, and ammonium persulfate, were prepared and standardized as described previously. 393 Investigation of Pd 2+ signaling with Res-DT We investigated the Pd 2+ sensing behavior of Res-DT in a pH 7.4 phosphate-buffered saline (PBS) solution, using 2% DMSO as a solubilizer.To that end, the analyte stock solution (15 mL, 5.0 mM) was rst added to a sample tube and diluted with DI water (2.34 mL) and a predetermined amount of DMSO.Then, a PBS solution (0.30 mL, 100 mM) and SDS (0.30 mL, 100 mM) were added to the mixture, followed by Res-DT (30 mL, 0.50 mM).The nal concentrations of Res-DT, the analyte, PBS, and SDS were 5.0 mM, 25 mM, 10.0 mM, and 10.0 mM respectively.Error bars were determined based on the standard deviation derived from three sets of experiments.

Mechanism study of Pd 2+ signaling
In a 100 mL round bottom ask, Res-DT (37 mg, 100 mmol) was dissolved in CH 3 CN (20 mL).The resulting solution was then mixed with palladium acetate (56 mg, 250 mmol).Upon verifying the completion of the reaction by thin-layer chromatography, the precipitate was collected using a centrifuge (4000 rpm) and washed with CH 3 CN.The precipitate was dried and analyzed using eld-emission scanning electron microscopy (FE-SEM) equipped with energy-dispersive X-ray spectroscopy (EDX).
The remaining solution was evaporated under reduced pressure and the residue was puried by column chromatography.The puried product of the Pd 2+ signaling of Res-DT was scrutinized by 1 H NMR and mass spectrometry measurements.

Office scanner-based determination of Pd 2+ concentration in Pd-containing catalyst and drug candidate
To determine the Pd 2+ concentration in Pd-containing catalyst and drug candidate, ve samples with varying Pd 2+ levels were tested.These samples were prepared by mixing a predetermined amount of the Pd-containing catalyst or drug (ranging from 0 to 6 mL; 1.0 mM), DI water (2.34 mL), DMSO (15 mL), PBS (0.30 mL, 0.10 M), SDS (0.30 mL, 0.10 M), and Res-DT (30 mL, 0.50 mM) in a sample vial.The resulting solutions (0.30 mL) were then individually transferred into a 96-well plate.A calibration plot of the color channel levels (RGB) against the Pd species concentration was constructed thereaer using an office scanner (V550, Epson) in transmittance mode.

Results and discussion
Sulfur-based chemosignaling probes have been extensively used to detect thiophilic metal ions and common oxidants through the desulfurization and oxidative hydrolysis reactions. 38,40In the present study, the dithioate moiety, which is prone to desulfurization-induced hydrolysis in the presence of hypochlorite ions, 38 was targeted in this study to develop a colorimetric, uorescent signaling probe for analyzing Pd 2+ in catalysts and drugs.The undesired response toward hypochlorite ions can be readily suppressed using hypochloritescavenging DMSO. 41Based on this rationale, a simple but optically vibrant dithioate-based Pd 2+ signaling probe, called Res-DT, was developed.Probe Res-DT was synthesized by reacting resorun with phenyl chlorodithioformate (Scheme 1) and then characterized by NMR spectroscopy and mass spectrometry.
First, the Pd 2+ signaling condition of Res-DT was optimized by measuring the changes in absorbance at 572 nm.Preliminary results indicated that Res-DT exhibited moderate Pd 2+ signaling activity in pH 7.4 PBS with 2% DMSO which acted both as a solubilizer and a scavenger for potential hypochlorite interferants (Fig. S1, ESI †).However, the signaling performance was relatively sluggish for practical applications, given that the reaction was incomplete even aer 30 min.Therefore, to improve the reaction rate of Res-DT for Pd 2+ ions, three types of surfactants were employed: anionic (SDS), cationic (cetyltrimethylammonium bromide (CTAB)), and nonionic (Tween 20).According to the results (Fig. S2a, ESI †), the Pd 2+ signaling behavior of Res-DT was substantially improved upon using SDS (10.0 mM).The other surfactant solutions (CTAB and Tween 20) also enhanced the Pd 2+ signaling speed of Res-DT (Fig. S2b and  c, ESI †).However, Res-DT was slightly hydrolyzed when the CTAB solution (1.0 mM) was used, and undesirable precipitates were formed when the Tween 20-containing solution (0.09 mM) was employed (Fig. S3, ESI †).Therefore, subsequent Pd 2+ signaling experiments with Res-DT were performed using the SDS surfactant solution as a signal booster.
The colorimetric and uorescence signaling properties of Res-DT for representative metal ions and anions were investigated under optimized conditions.First, the colorimetric signaling response of Res-DT was examined upon exposure to various common metal ions (Fig. 1).Res-DT exhibited a weak absorption band at 452 nm with pale-yellow coloration in the measurement solution.However, aer treatment with Pd 2+ ions, it showed a strong increase and moderate decrease in the absorbance at 572 and 452 nm, respectively.Concomitantly, the solution turned magenta from pale yellow (Fig. 1, inset).Furthermore, the other tested metal ions did not induce noticeable changes in the UV-vis spectra or color.Because the signaling ability of Res-DT for Pd 2+ was related to the absorbance variation at two widely separated wavelengths, 572 nm and 452 nm, the selectivity for Pd 2+ was assessed by ratiometry using the absorbance ratio (A 572 /A 452 ) (Fig. 1).The A 572 /A 452 value of Res-DT with Pd 2+ was 10.2, whereas those of the other tested metal ions were remarkably low and similar to that of Res-DT alone (values ranging from 0.04 (Mg 2+ ) to 0.11 (Pt 2+ )).Additionally, the Pd 2+ selectivity of Res-DT was also conrmed in the presence of several representative anions (Fig. S4, ESI † )).Furthermore, taking into the fact that the dithioate-based probe has been used for hypochlorite sensing, 38 the changes in the absorbance ratio of Res-DT upon treatment with representative oxidants were measured.The results (Fig. S5, ESI †) indicated that the tested oxidants, including hypochlorite ions, negligibly altered the A 572 /A 452 ratio (ranging from 0.05 for perborate (PB) to 0.06 for tert-butyl hydroperoxide (TBHP)).The photophysical properties of Res-DT, both pre-and post-Pd 2+ signaling, are detailed in Table S2 (ESI).† Next, to conrm the inuence of background ions on the Pd 2+ signaling tendency of Res-DT, sensing experiments with coexisting metal ions and anions were performed.According to the results (Fig. 2), the Pd 2+ signaling behavior of Res-DT was unaffected by the presence of coexisting metal ions.Essentially, the A 572 /A 452 value of the samples aer the Pd 2+ signaling experiments varied only slightly, ranging from 93.6% (for Fe 2+ ) to 107.3% (for Ba 2+ ) of the control result.In addition, the Pd 2+ signaling performance of Res-DT was not altered by the Scheme 1 Preparation of the Pd 2+ signaling probe (Res-DT).Resorun-based sensing probes typically exhibit signicant uorescence features as well as remarkable colorimetric signaling behavior. 42Therefore, the Pd 2+ signaling performance of Res-DT was evaluated based on the changes in the uorescence response occurring under the same sensing conditions.Based on the results (Fig. 3, inset), Res-DT exhibited faint uorescence emission at approximately 586 nm (F Res-DT = 0.009) owing to the photophysical characteristics of the phenolic moiety-protected resorun uorophore. 43However, upon exposure to Pd 2+ ions, Res-DT revealed strong uorescence emission (F Res-DT+Pd(II) = 0.58), exhibiting over a 90-fold uorescence enhancement at 591 nm (Fig. 3).All other tested metal ions showed insignicant uorescence responses except for Au 3+ ions (I/I 0 = 5.88), with I/I 0 generally uctuating between 0.97 (for Li + ) and 1.32 (for Pt 2+ ).Furthermore, no measurable changes were observed in the uorescence emission of Res-DT toward the encountered anions, with I/I 0 varying from 0.99 (for Cl − ) to 1.50 (for N 3 − ) (Fig. S7, ESI †).These results highlight the uorometric potential of Res-DT to sense Pd 2+ ions in chemical and industrial applications.However, the Pd 2+ sensing behavior of Res-DT was investigated using colorimetric measurements which allowed the ratiometric analysis, rather than the method relying on a simple turn-on type uorescence enhancement at a single wavelength.Ratiometry offers several advantages including increased sensitivity, improved selectivity, and reduced interference from the effects of interfering substances in the sample matrix. 44he Pd 2+ signaling was hypothesized to be caused by the generation of the resorun uorochrome via Pd 2+ -mediated hydrolysis of the dithioate moiety of Res-DT (Scheme 2).In the proposed sensing mechanism of Res-DT, the initial stage involves complex formation between the sulfur atom of the C]S bond and thiophilic Pd 2+ ions. 45The complex is then hydrolyzed, yielding resorun dye with its distinctive magenta color and strong uorescence signals.To conrm the sensing mechanism of Res-DT, the Pd 2+ signaling product was scrutinized using 1 H NMR measurements.The results indicated that the NMR pattern of Res-DT was similar to that of typical phenolprotected resorun compounds (Fig. 4). 40Moreover, the puri-ed Pd 2+ sensing product (Res-DT + Pd 2+ ) exhibited three well-dened resonances in the 1 H NMR spectrum that were likely associated with resorun.Furthermore, our investigation using thin-layer chromatography of the Pd 2+ signaling solution of Res-DT revealed that resorun is produced (Fig. S8, ESI †).Through FAB mass spectrometry, a diagnostic peak at m/z = 214 was identied in the Pd 2+ signaling product, which matches the calculated mass of resorun (C 12 H 8 NO 3 + [M + H] = 214) (Fig. S9, ESI †).Additionally, we isolated a greenish-black colored precipitate from the Pd 2+ signaling solution and conrmed it to be PdS using FE-SEM equipped with EDX (Fig. S10, ESI †).
The inuence of pH on the Pd 2+ signaling performance of Res-DT was tested to assess its practical usability.The results showed that the A 572 /A 452 value of pristine Res-DT remained constant across the pH range 4.0-10.0(Fig. 5).In contrast, that of the Pd 2+ signaling sample (Res-DT + Pd 2+ ) increased signicantly from pH 6.0 onward and stabilized at around pH 8; this tendency was mirrored by that of the reference compound resorun in the presence of coexisting Pd 2+ ions.This nding  UV-vis titration of Res-DT with Pd 2+ was subsequently performed to determine the minimum Pd 2+ ion concentration detected using Res-DT.The absorbance ratio A 572 /A 452 increased linearly up to a Pd 2+ concentration of 5.0 mM (R 2 = 0.9987) (Fig. 6).Using the titration plot and IUPAC recommended equation (3s blk / m), where s blk and m denote standard deviation of the blank signal and analytical sensitivity, respectively, the detection limit was calculated to be 10 nM. 46Additionally, we studied the quantitative analytical behavior of Res-DT for Pd 2+ sensing by uorescence titration (Fig. S12, ESI †).The uorescence emission changes at 591 nm demonstrated a linear correlation with increasing concentrations of Pd 2+ (R 2 = 0.9870).From these results, the detection limit for Pd 2+ ions was calculated to be 7.3 nM. 46urthermore, we estimated the detection time of Res-DT for Pd 2+ ions by observing the time-dependent change in the absorbance ratio (A 572 /A 452 ), and it was found to be 7 min (Fig. S13, ESI †).
Finally, to assess the practical applicability of the devised probe, Pd assay in Pd-containing catalyst and drug candidate was performed using an office scanner as a readily accessible device for detection. 47We used the white catalyst as the Pdcontaining catalyst, and a Pd 2+ -2-picolinic acid complex as the Pd-containing drug candidate. 48The Res-DT with the tested catalyst and drug candidate exhibited a noticeable color shi from yellow to magenta, and the variation in color could be conveniently characterized through analysis of the RGB color channel levels of the scanned image (Fig. 7 and S14, ESI †).Consequently, acceptable calibration curves based on the green channel, rather than the red and blue channels, were obtained for the catalyst and drug candidate, which yielded satisfactory R 2 values of 0.9732 and 0.9887 for the catalyst and drug candidate, respectively.The results of the colorimetric signalingbased Pd 2+ assay for the catalyst and drug candidate performed using the office scanner (recovery = 94.1-107.2%)were consistent with those of UV-vis spectrometry (recovery = 91.0-101.3%)(Table S3, ESI †).This result implies that the Res-DT could be successfully applied to the analysis of Pd 2+ ions in palladium relevant catalysts and drugs.

Conclusions
A simple colorimetric and uorescent probe (Res-DT) was developed for the convenient determination of Pd species in Pd-   containing catalyst and drug candidate.Res-DT exhibited selective, sensitive signaling behavior toward Pd species without interference from common metal ions, anions, and oxidants.The signaling was achieved by Pd-induced hydrolysis of the dithioate moiety of Res-DT, generating resorun dye that exhibits a magenta color, visible to the naked eye, and intense uorescence.The probe was immune to interference from several other metal ions and anions when detecting Pd ions.Additionally, the Pd signaling was achieved within 7 min, and the detection limit of the probe was determined to be 10 nM.Finally, Res-DT was successfully employed to analyze Pd 2+ ions in Pd-containing catalyst and drug candidate.The designed Res-DT can potentially be applied in various practical and industrial settings featuring Pd-related systems.
) by showing the low A 572 /A 452 values of Res-DT for anions (ranging from 0.06 (Cl − ) to 0.08 (N 3 −