A Novel Pyrene-Based Fluorescent Probe for the Detection of Cu²⁺

Abstract

A novel fluorescent probe (PYB) for selective and sensitive detection of Cu²⁺ ions was rationally designed and synthesized via a multi-step organic reaction using pyrene as the fluorophore and salicylaldehyde-diethylenetriamine Schiff base as the recognition moiety. The structural characterization of PYB was confirmed by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry (HRMS). Photophysical properties investigation revealed that the probe exhibited strong fluorescence emission at 362 nm in DMF/HEPES-NaOH buffer solution (v:v = 1:1, pH 7.4), which underwent a significant fluorescence quenching response (quenching efficiency up to 77%) upon the addition of Cu²⁺, attributed to the formation of a 1:1 PYB-Cu²⁺ complex (binding constant K = 799.65 M⁻¹). The probe showed excellent selectivity for Cu²⁺ over other common metal ions (Ba²⁺, Na⁺, Mg²⁺, Zn²⁺, Cd²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Hg²⁺, Fe³⁺, Co²⁺), with a low detection limit of 8.35 × 10⁻⁷ M, which is well below the maximum allowable concentration of Cu²⁺ in drinking water specified by the World Health Organization (WHO). Furthermore, a portable fluorescent test strip based on PYB was successfully fabricated, enabling rapid and visual detection of Cu²⁺ under UV light. Fluorescence imaging experiments in living HepG2 cells demonstrated that PYB could penetrate cell membranes efficiently and realize the intracellular detection of exogenous Cu²⁺. These results collectively indicate that PYB holds great potential as a practical tool for Cu²⁺ detection in environmental monitoring, food safety, and biological systems.

1. Introduction

Copper ions (Cu²⁺) are essential trace elements in living organisms and exert crucial regulatory effects on various physiological and metabolic processes through multiple mechanisms [1,2,3]. As key cofactors for numerous enzymes, including cytochrome oxidase, ceruloplasmin, and copper-zinc superoxide dismutase (Cu/Zn-SOD), Cu²⁺ participates in core biological events such as energy metabolism, redox balance, and material synthesis [4,5,6]. Specifically, cytochrome oxidase mediates electron transfer in the mitochondrial respiratory chain, which is indispensable for oxidative phosphorylation and ATP production [4]; ceruloplasmin catalyzes the oxidation of ferrous ions (Fe²⁺) to ferric ions (Fe³⁺) in serum, thereby promoting iron binding to transferrin and regulating systemic iron homeostasis [5]; and Cu/Zn-SOD scavenges intracellular superoxide anions (O₂⁻·) through dismutation, mitigating oxidative stress and protecting cells from oxidative damage [6]. Beyond enzymatic catalysis, Cu²⁺ modulates diverse metabolic pathways; it influences glucose metabolism by regulating the balance of insulin and glucagon [7], participates in lipid synthesis and decomposition, and indirectly affects hemoglobin and protein biosynthesis via regulating iron absorption and transport [8]. Deficiency of Cu²⁺ may lead to hypercholesterolemia, cardiovascular diseases, and bone mineralization disorders, while excessive Cu²⁺ accumulation can induce hepatotoxicity, neurotoxicity, and even life-threatening conditions [9,10]. Additionally, Cu²⁺ plays a pivotal role in immune function regulation by modulating the activity of T lymphocytes and B lymphocytes and the secretion of pro-inflammatory cytokines (e.g., TNF-α and IL-6) [11,12,13], and is involved in the biosynthesis of neurotransmitters such as dopamine and catecholamine in the central nervous system, safeguarding nerve cell integrity [14,15,16]. The maintenance of Cu²⁺ homeostasis relies on precise absorption (primarily in small intestinal epithelial cells) and excretion (predominantly via bile, with minor elimination through feces and urine) [17,18,19,20]. Given the dual role of Cu²⁺ as an essential nutrient and a potential toxin, the development of efficient, selective, and sensitive methods for Cu²⁺ detection is of great significance for environmental monitoring, food safety, and biomedical research.

Fluorescent probes have emerged as a powerful tool for metal ion detection due to their advantages of high sensitivity, rapid response, non-invasiveness, and real-time visualization [21,22,23]. Among various fluorophores, pyrene and its derivatives have garnered extensive attention in the design of fluorescent probes, attributed to their unique photophysical properties: strong fluorescence quantum yield, long fluorescence lifetime, characteristic monomer/excimer emission peaks (typically around 375–395 nm for monomers and 470 nm for excimers), and excellent photostability [14]. These features enable pyrene-based probes to achieve signal transduction through mechanisms such as photoinduced electron transfer (PET), intramolecular charge transfer (ICT), excimer formation/dissociation, and fluorescence resonance energy transfer (FRET), making them suitable for the detection of various analytes, including metal ions, anions, and biomolecules [24].

In recent years, numerous pyrene-derived fluorescent probes for Cu²⁺ detection have been reported. For example, pyrene Schiff base derivatives have been designed to recognize Cu²⁺ via PET quenching, exhibiting moderate sensitivity with detection limits in the range of 0.5–5 μmol·L⁻¹ [24]. Pyrene-based macrocyclic probes (e.g., crown ethers and cryptands) have shown improved selectivity but suffer from complex synthesis routes and poor water solubility [25]. Some pyrene-amide conjugates have achieved Cu²⁺ detection in aqueous media, but their fluorescence quenching efficiency is relatively low and their application in biological systems is limited due to inadequate cell membrane permeability [26]. Despite these advances, existing pyrene-derived Cu²⁺ probes still face challenges such as insufficient water solubility, low quenching efficiency, poor biocompatibility, or complicated synthesis processes, which hinder their practical application in environmental and biological samples [27].

To address these limitations, herein we designed and synthesized a novel pyrene-based fluorescent probe (PYB) by integrating pyrene as the fluorophore and salicylaldehyde-diethylenetriamine Schiff base as the Cu²⁺ recognition moiety. The Schiff base moiety was selected for its strong chelating ability with Cu²⁺, while the pyrene fluorophore ensured excellent photophysical properties. Compared with previously reported pyrene-derived Cu²⁺ probes, PYB exhibits high fluorescence quenching efficiency, and a low detection limit. Furthermore, the probe demonstrates negligible cytotoxicity and efficient cell membrane penetration, enabling intracellular Cu²⁺ imaging. Additionally, a portable fluorescent test strip based on PYB was fabricated for rapid and visual Cu²⁺ detection. Collectively, this work provides a promising tool for Cu²⁺ detection in environmental and biological systems, expanding the application scope of pyrene-based fluorescent probes.

2. Materials and Methods

2.1. Materials and Instruments

The chemicals and reagents involved in the study were purchased from commercial suppliers (Aladdin Reagent, Shanghai, China)and used without further purification. The solvents for spectra detection were HPLC (High Performance Liquid Chromatography Grad) reagents without fluorescent impurity. Solutions of different ions (Cu²⁺, Ca²⁺, Ba²⁺, Hg²⁺, Fe³⁺, Mn²⁺, Na⁺, Mg²⁺, Co²⁺, Cd²⁺, Zn²⁺, Pb²⁺) in titration experiments were from CuCl₂, CrCl₃·6H₂O, CaCl₂, FeCl₂, Hg(NO₃)₂, FeCl₃, Mn(NO₃)₂·6H₂O, NaNO₃, MgCl₂, CoCl₂, CdCl₂, ZnCl₂, Pb(NO₃)₂,(Aladdin Reagent, Shanghai, China) and dissolved in HEPES (N-2-Hydroxyethylpiperazine-N-2-Ethane Sulfonic Acid)-NaOH buffer solution at pH 7.4.

Experimental intermediate and Probe PYB were characterized by ¹H NMR (Nuclear Magnetic Resonance), ¹³C NMR (Varian mercury-300 spectrometer, Palo Alto, CA, USA) and HRMS (High Resolution Mass Spectrometry) analyses (Agilent 1290-micro TOF QII, Santa Clara, CA, USA). Relevant data are provided in the Supporting Information (Figures S1–S3). UV-vis spectra were obtained with a Shimadzu UV-2600 (Kyoto, Japan), using a quartz cuvette (d = 1 cm) in the wavelength range of 275–375 nm. The fluorescence spectra were obtained with a Hitachi F-4500 spectrofluorimeter (Tokyo, Japan), using quartz cell (d = 1 cm) in the wavelength range of 325–450 nm, the excitation and emission slits are both 5 nm. The test instrument for pH is Mettler–Toledo Instrument DELTE 320 pH (Parramatta, NSW, Australia). The fluorescent cell image experimental device consisted of an Olympus IX-70 fluorescence microscope and an Olympus c-5050 digital camera (Tokyo, Japan).

2.2. Synthesis of Probe PYB

Synthesis of Intermediate 1: Salicylaldehyde (2.6 g, 21 mmol, 98%, Aladdin Reagent, Shanghai, China) was dissolved in 100 mL of anhydrous ethanol (≥99.5%, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) under ambient temperature. Diethylenetriamine (1.0 g, 10 mmol, 99%) was added dropwise to the above homogeneous solution with continuous stirring over a period of 15 min, and the resulting mixture was stirred at room temperature (25 °C) for 2 h. Subsequently, the reaction system was heated to 75 °C in an oil bath and maintained at this temperature for an additional 2 h with magnetic stirring. After completion of the reaction (monitored by thin-layer chromatography, TLC), the solvent was removed under reduced pressure using a rotary evaporator to afford the crude product as a brownish viscous liquid. The crude product was purified by silica gel column chromatography (SiO₂, 200–300 mesh) using dichloromethane/ethanol (v/v = 10:1) as the eluent. Intermediate 1 was obtained as a pale yellow oil (2.34 g) with a yield of 75.0%. ¹H NMR (300 MHz DMSO-d6, 25 °C, TMS) δ: 11.11 (s, 2H, -OH). 8.50 (s, 2H, -CH=), 7.77–7.76 (d, 2H, Ar-H), 7.34–7.32 (m, 2H, Ar-H), 7.07–6.96 (m, 2H, Ar-H), 6.77–6.76 (d, 2H, Ar-H), 3.76 (d, 4H, -CH₂-), 2.87 (d, 4H, -CH₂-), 1.50 (s, H, -NH-). ¹³C NMR (75 MHz DMSO-d6, 25 °C, TMS) δ: 163.30, 158.01, 132.22, 127.58, 122.26, 116.54, 54.91, 49.95. (Figures S1 and S2).

Synthesis of Intermediate 2: 1-Pyrenemethanol (0.232 g, 1 mmol, 97%, Aladdin Reagent, Shanghai, China), 4-bromobutyric acid (0.184 g, 1 mmol, 98%, Aladdin Reagent, Shanghai, China), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 0.335 g, 1.75 mmol, 99%, Aladdin Reagent, Shanghai, China) and 4-dimethylaminopyridine (DMAP, 0.012 g, 0.1 mmol, 99%, Aladdin Reagent, Shanghai, China) were sequentially added to a 50 mL round-bottomed flask equipped with a magnetic stir bar. Anhydrous dichloromethane (20 mL, ≥99.8%) was added to dissolve the reactants, and the flask was sealed with a rubber stopper under atmospheric conditions. The reaction mixture was stirred magnetically at room temperature (25 °C) for 15 h. After TLC confirmation of complete conversion, the solvent was evaporated under reduced pressure to yield the crude product. Purification was performed by silica gel column chromatography (SiO₂, 200–300 mesh) using petroleum ether/ethyl acetate (v/v = 2:1) as the eluent, affording Intermediate 2 as a bright yellow oil (0.25 g) with a yield of 65.4%. ¹H NMR (300 MHz DMSO-d6, 25°C, TMS) δ: 8.37–7.66 (m, 9H, Ar-H), 5.50 (s, 2H, -CH₂-), 3.51 (d, 2H, -CH₂-), 2.32 (d, 2H, -CH₂-), 2.16 (d,2H,-CH₂-). ¹³C NMR (75 MHz DMSO-d6, 25 °C, TMS) δ: 173.36, 139.60, 133.69, 132.23, 126.11, 125.65, 125.23, 124.45, 123.69, 123.29, 122.55, 122.27, 121.82, 65.98, 33.29, 32.55, 27.53. (Figures S3 and S4).

Potassium carbonate (K₂CO₃, 1.40 g, anhydrous, 99%), potassium iodide (KI, 0.07 g, 99%, used as a catalyst), Intermediate 1 (0.23 g, 0.75 mmol) and Intermediate 2 (0.19 g, 0.50 mmol) were placed in a 100 mL three-necked flask fitted with a reflux condenser and a nitrogen inlet. Anhydrous acetonitrile (50 mL, ≥99.5%) was added as the solvent, and the reaction system was purged with high-purity nitrogen for 30 min to remove oxygen. The mixture was heated to reflux temperature (82 °C) under nitrogen atmosphere and stirred magnetically for 6 h. After the reaction was completed (monitored by TLC), the solvent was removed under reduced pressure to obtain crude product. The crude product was purified by silica gel column chromatography (SiO₂, 200–300 mesh) using dichloromethane/ethanol (v/v = 10:1) as the eluent. The target Probe PYB, pyren-1-ylmethyl 4-(bis(2-(((E)-2-hydroxybenzylidene)amino)ethyl)amino) butanoate, was obtained as a yellow viscous solid (0.34 g) with a yield of 55.5%. The detailed synthetic route is illustrated in Scheme 1. ¹H NMR (300 MHz DMSO-d6, 25 °C, TMS) δ: 11.13 (s, 2H, -OH).8.49 (d, 2H, -CH=), 7.86–7.71 (m, 6H, Ar-H), 7.59–7.41 (m, 5H, Ar-H), 7.25–7.12 (m, 4H, Ar-H), 6.78–6.77 (m, 2H, Ar-H), 5.30 (m, 2H, -CH₂-), 3.81–3.80 (d, 4H,-CH₂-), 2.94–2.90 (d, 4H, -CH₂-), 2.52 (s, 2H, -CH₂-), 2.48 (s, 2H, -CH₂-), 1.76–1.72 (d,2H,-CH₂-). ¹³C NMR (75 MHz DMSO-d6, 25 °C, TMS) δ: 176.79, 161.79, 158.76, 139.53, 133.07, 132.59, 131.82, 126.29, 124.53, 124.11, 123.29, 122.82, 121.86, 121.38, 119.72, 119.38, 114.07, 113.18, 63.18, 61.07, 59.38, 51.98, 32.00, 21.82. ESI-MS m/z [M]⁺ calc. 611.3, obs. 611.7. (Figures S5–S7).

3. Results

3.1. Absorption Spectral Response

The UV-vis absorption spectra of Probe PYB in different concentrations of Cu²⁺ (1 × 10⁻⁴ mol/L–5 × 10⁻⁴ mol/L) are illustrated in Figure 1. Probe PYB was dissolved in a DMF(N,N-Dimethylformamide)/HEPES-NaOH buffer solution (5 × 10⁻⁴ mol/L, v:v = 1:1, pH 7.4) solution. The maximum absorption peak of blank Probe PYB appeared at 320 nm. Under the same testing environment, different concentrations of Cu²⁺ were added to the solution of probe PYB. The absorbance at 320 nm gradually enhanced with the increase of Cu²⁺ concentration. Therefore, in the subsequent determination of fluorescence emission spectra, 320 nm was used as the maximum excitation wavelength.

3.2. Fluorescence Spectral Response

To clarify the sensitivity of Probe PYB to Cu²⁺, the fluorescence spectra of Probe PYB in different concentrations of Cu²⁺ (5 × 10⁻⁵ mol/L–5 × 10⁻⁴ mol/L) were tested (V_DMF:V_{HEPES-NaOH buffer solution} = 1:1, pH 7.4, λ_ex = 320, λ_em = 362). As illustrated in Figure 2, it could be clearly observed that the fluorescence intensity of Probe PYB gradually decreased with the increase of Cu²⁺ concentration. When the molar ratio of the Probe PYB to Cu²⁺ reached 1:1, the fluorescence quenching rate was approximately 71%. By measuring the relationship between the maximum fluorescence emission intensities (362 nm) and Cu²⁺ concentrations, it was determined that this fluorescence quenching phenomenon conforms to a linear relationship (y = 496.71394x + 1449.69333, R² = 0.99145), indicating that Probe PYB can perform quantitative analysis on Cu²⁺ and verifying its good sensitivity to Cu²⁺. And based on the detection limit formula (DL = 3 s/m, where s is the standard deviation of replication measurements, and m is the slope of the calibration curve), the detection limit of Probe PYB is calculated to be 8.35 × 10⁻⁷ M [21].

Selectivity is an important indicator for measuring the quality of fluorescent probes. In order to verify the selectivity of Probe PYB for different metal ions, several common metal ions (Ba²⁺, Na⁺, Mg²⁺, Zn²⁺, Cd²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Cu²⁺, Hg²⁺, Fe³⁺, Co²⁺, 5 × 10⁻⁴ mol/L) were added to the Probe PYB (5 × 10⁻⁴ mol/L) solution under the same measurement conditions (V_DMF:V_buffer = 1:1, pH 7.4) and their fluorescence intensities were tested separately. As illustrated in Figure 3, the purple bar represented the maximum fluorescence emission intensities of Probe PYB in different metal ions (λ_ex = 320 nm, λ_em = 362 nm). It could be obviously determined that only Cu²⁺ caused a significant decrease in the fluorescence intensity of Probe PYB, while other metal ions would not have a significant impact. To further verify the detection ability of Probe PYB for Cu²⁺, in complex environments, we also introduced competitive experiments as a reference. After inducing quenching of Probe PYB (5 × 10⁻⁴ mol/L) with Cu²⁺ (5 × 10⁻⁴ mol/L), 10 times the concentration of other metal ions (Ba²⁺, Na⁺, Mg²⁺, Zn²⁺, Cd²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Hg²⁺, Fe³⁺, Co²⁺, 5 × 10⁻³ mol/L) were added and the fluorescence intensity of the system was measured (V_DMF:V_{HEPES-NaOH buffer solution} = 1:1, pH 7.4). The maximum fluorescence intensities at 362 nm were selected as experimental data. As shown in Figure 3, the green bar represents the competitive experiments. It could be found that the introduction of 10 equivalents of other metal ions did not significantly affect Cu²⁺ recognition based on fluorescence quenching. This meant that Probe PYB could still effectively recognize Cu²⁺ in the presence of high concentrations of interfering ions, and also verifies Probe PYB’s good qualitative analysis ability for Cu²⁺.

Moreover, the quenching rates of Probe PYB in above-mentioned ions were also measured. As illustrated in Figure 4, the quenching rate of Probe PYB induced by Cu²⁺ is very prominent, reaching 77%, which is significantly better than other metal ion.

$$\lg ({\displaystyle \frac{I_0-I}{I}})=\lg K_{SV}+\mathrm{nlg}(Q)$$

(1)

The Stern–Volmer equation (Equation (1)) was introduced to calculate the quenching constant and binding ratio of Probe PYB to Cu²⁺ [23]. Wherein, I₀ is the fluorescence intensity of Probe PYB, I is the fluorescence intensity of Probe PYB induced by Cu²⁺, K_SV is the Stern Volmer constant, n is the number of binding sites, and Q is the concentration of Cu²⁺. Based on the fluorescence intensity of Probe PYB under the condition of Cu²⁺ concentration from 5 × 10⁻⁵ M to 5 × 10⁻⁴ M, the equation is calculated to be y = 0.92582x + 2.9029. By calculating the slope of the equation, n is equal to 0.92, approximately equal to 1, indicating that the coordination ratio between Cu²⁺ and Probe PYB is 1:1. And according to the intercept of the equation, the quenching constant can be calculated to be 799.65 M⁻¹. Based on the coordination atoms present in the probe molecule and relevant literature reports, we have plotted the coordination model between Probe PYB and Cu²⁺ in Figure 5.

3.3. Test Strip Experiment

In addition, filter paper was selected as the carrier for Probe PYB for convenient and rapid detection of Cu²⁺. The test strip for detecting copper ions was prepared by uniformly dropping Probe PYB solution onto neutral filter paper and drying it. Subsequently, solutions of the above-mentioned metal ions were dropped onto the surface of the test paper and placed under the irradiation of ultraviolet light, as shown in Figure 6, only Cu²⁺ induced fluorescence quenching of the test strip, while other metal ions did not significantly affect the fluorescence signal of the test strip.

3.4. Biological Investigation

In order to expand the application of Probe PYB in the field of biology, the biocompatibility of Sensor PYB was assessed in HepG2 cells. The MTT assay was performed to confirm the cytotoxicity in different concentrations of Sensor PYB (0.1–10 µM). As illustrated in Figure 7, the cell viability had no dramatic differences within the concentration range of 0.1–5 µM, when the concentration of Sensor PYB reached 10 µM or above, cell viability began to decrease. Even so, at the highest concentration of 100 µM, the cells maintained fine viability (59%), and the IC₅₀ value was 171.5 µM. So the cytotoxicity of Sensor PYB was relatively low under the experimental conditions.

Furthermore, we conducted fluorescence cell imaging experiments on HepG2 cells using Probe PYB. The pre-cultured HepG2 cells were removed from the incubator, the cell culture medium was aspirated using a micropipette and washed 3 times with phosphate-buffer solution (pH = 7.4) to ensure removal of the medium. Then added Probe PYB solution (1 × 10⁻⁵ M in DMSO) to the cell culture plate, and cultivated for 30 min at room temperature to ensure Probe PYB completely entered the cells. Afterwards, washed the cells 3 times with buffer solution and transferred to a fluorescence microscope for observation. As shown in Figure 8a, the cell outline was clear and complete, indicating that Probe PYB did not cause damage to the cells. From Figure 8b, it could be observed that the cells treated with Probe PYB exhibited significant fluorescence emission, indicating that the probe could penetrate the cell membrane and be adsorbed by the cells. Subsequently, 1 × 10⁻⁵ M Cu²⁺ buffer solution (pH 7.4) was added to the cells and cultured for 30 min. The corresponding fluorescence imaging is shown in Figure 8c. After interacting with Cu²⁺, the fluorescence of cells treated with Probe PYB significantly disappeared, indicating that Probe PYB coordinated with Cu²⁺ inside the cell and demonstrating its good biocompatibility.

To further validate the fluorescence imaging ability of Probe PYB, zebrafish was selected as the test sample for the experiment. Zebrafish is a highly dynamic tropical fish with a body length of about 3 cm and a genetic similarity of up to 87% to humans. First, adult zebrafish was cultivated in distilled water containing 20% Probe PYB (10 µM) in DMSO solution for 1 hour. Then transferred the zebrafish to distilled water and washed it three times with buffer solution (pH 7.4) before conducting fluorescence imaging experiments. As illustrated in Figure 9a, the zebrafish cultured in Probe PYB solution showed fluorescence under UV light irradiation, with clear contours, indicating that Probe PYB had been successfully adsorbed. Afterward, transferred the zebrafish to 1 × 10⁻⁵ M Cu²⁺ buffer solution (pH 7.4) for 1 hour of cultivation, washed it three times with deionized water, and then performed fluorescence imaging experiments. As shown in Figure 9b, under UV light irradiation, the fluorescence on the surface of zebrafish was quenched after treatment with Cu²⁺ solution. This phenomenon once again demonstrated the biocompatibility of Probe PYB and proved its practical value in the field of biology.

4. Conclusions

In this study, a novel Cu²⁺ fluorescent probe was successfully designed and synthesized based on a pyrene derivative. Through titration experiments, Probe PYB exhibits good selectivity and sensitivity toward Cu²⁺, and it still retains the ability to recognize Cu²⁺ in the presence of high concentrations of interfering ions. The detection limit of Probe PYB is calculated to be 8.35 × 10⁻⁷ M based on the detection limit formula, and the complexation constant between Probe PYB and Cu²⁺ is calculated to be 799.65 M⁻¹ in a 1:1 binding mode based on the Stern–Volmer equation. The ability of Probe PYB to quickly and conveniently detect Cu²⁺ is verified by preparing fluorescent test strips soaked with probe solution. Moreover, Probe PYB has been successfully applied in live-cell fluorescence imaging and biological imaging, thus demonstrating its value in the field of fluorescence tracing.