A spiropyran-based colorimetric probe for the highly selective and sensitive detection of copper(II) ions

Abstract

A highly selective, sensitive and convenient probe, based on a photochromic and acidochromic benzoxazole-spiropyran derivative, was developed for visual Cu²⁺ detection. The detection remained unaffected by competing ions and achieved a detection limit of 10⁻⁷ M in both solution and test strip formats.

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Trace copper ions, particularly the divalent Cu²⁺ are crucial for biological systems. Copper serves as an essential catalytic and structural cofactor in numerous life processes,¹ including cellular respiration,² antioxidant defense,³ neurotransmitter synthesis,⁴ connective tissue formation,⁵ and iron metabolism.⁶ The human body maintains copper homeostasis through sophisticated import, export, and storage mechanisms. Disruptions in this balance can be catastrophic, as demonstrated by genetic disorders such as Wilson's disease (copper overload)⁷ and Menkes disease (copper deficiency).⁸

On the other hand, widespread copper applications, including industrial catalysts and agricultural fungicides, cause environmental pollution. This results in high toxicity towards aquatic life (particularly fish and invertebrates) even at low concentrations.⁹ Thus, highly sensitive analytical methods are needed.

Traditional copper analysis techniques, including atomic absorption spectroscopy (AAS),¹⁰ high-performance liquid chromatography (HPLC),¹¹ and inductively coupled plasma mass spectrometry (ICP-MS),¹² provide highly sensitive detection capabilities for multiple metal elements. However, these methods are cost-prohibitive and require sophisticated laboratory infrastructure as well as skilled personnel. In contrast, the development of sensors with highly selective and sensitive responses to specific metal ions represents a promising alternative. Similar to pH test strips used for measuring solution acidity, such sensors offer simplicity and ease of operation.

Spiropyrans and their derivatives, which are among the most widely studied photochromic compounds, have been extensively applied in fields such as responsive materials,¹³ drug delivery,¹⁴ and cell adhesion.¹⁵ Upon exposure to UV light or binding with metal ions, the colorless spiropyran (SP) form isomerizes into the colored merocyanine (MC) form. This ring-opening reaction alters the electronic structure of the molecule, causing a distinct color change that is visible to the naked eye.¹⁶ Consequently, numerous spiropyran-based sensing platforms have been developed.¹⁷

Peng et al.¹⁸ recently reviewed spiropyrans functionalized with chelating agents (amines, carboxylic acids, macrocycles, heterocycles, organic dyes, copolymers, and nanoparticles), demonstrating their utility as fluorescent/colorimetric sensors for metal ions and anions in aqueous and biological systems. However, key limitations persist: (1) most sensors require improvement in metal ion selectivity; (2) many reported systems lack practical applicability; (3) SP–metal complex structures and mechanisms remain ambiguous. To address these challenges, we successfully synthesized a colorimetric spiropyran-based probe via a condensation reaction of 5-carboxy-1,2,3,3-tetramethyl-3H-indol and 3-benzoxazolyl-5-methyl-salicylaldehyde (Fig. 1 and Scheme S1). Interestingly, this probe exhibited high selectivity toward Cu²⁺ and strong anti-interference capability against other metal ions, achieving a detection limit of 10⁻⁷ M in both solution and test strip formats, thereby enabling convenient naked-eye detection of Cu²⁺.

Fig. 1 The schematic benzoxazole-spiropyran probe for highly selective, sensitive and convenient detection of Cu²⁺ by naked-eye.

The benzoxazole-spiropyran probe exhibited excellent photochromic response at low temperatures. As shown in Fig. 2A, the colorless SP methanolic solution readily turned cyan upon 365 nm irradiation at −40 °C, indicating that the closed SP convert to the open MC. When the UV light was removed, the MC structure would recover its original closed SP gradually. In this process, if exposed in natural light or heated to room temperature, the recovery would be accelerated. The UV-vis spectra (Fig. 2B) further confirmed such photochromic conversion. Before 365 nm irradiation, two absorption bands appeared at 280 and 346 nm, respectively. The former was ascribed to the π–π* transition of the benzoindoline ring in the closed spiropyran, while the latter was attributed to the π–π* transition of the benzopyran moiety.¹⁹ After 365 nm irradiation, two new absorption bands appeared at 426 and 645 nm, which were attributed to the π–π* transitions of two types of trans-MC forms (MC1 and MC2, the cis- or trans- was identified by the double bond between benzoindoline and phenoxide ring),²⁰ as illustrated in Fig. 2A. We speculated that initially formed MC1 readily isomerized to MC2 due to the higher acidity of the carboxyl group than the benzothiazole phenol. However, when the irradiation was at room temperature, no significant photochromic response occurred.

Fig. 2 (A) Photochromic response of the benzoxazole-spiropyran (SP) upon irradiation of 365 nm at −40 °C. Schematic reversible transition from phenolate (MC1) to carboxylate (MC2) of the ring-opened merocyanine isomers. And acidochromic response upon different pH values at room temperature. (B) UV-vis spectra of SP in methanol before and after 365 nm irradiation at −40 °C. (C) UV-vis spectra of the SP at different pH values. (D) ¹H NMR spectra of SP in DMSO-d6 with DCl at room temperature.

The introduction of H⁺ ions, which can protonate the MC benzofuran anions, was conducive to investigation of the SP → MC isomerization. Fig. 2A showed the acidochromic response of the benzoxazole-SP probe. The results indicated that the colorless SP solution turned to orange upon addition of H⁺ ions. And the UV-vis spectra (Fig. 2C) displayed that a distinct absorption at 458 nm appeared as pH < 3,²¹ confirming the formation of trans-MCH⁺ (Fig. 2A) in such isomerization. Compared to the absorption of the MC2 isomers (Fig. 2B), the absorption at 645 nm disappeared completely, revealing that only one isomer, MCH⁺, formed.

To further confirm the structure of the SP and MC isomers, ¹H NMR was employed. As shown in Fig. 2D, the ¹H NMR spectra of SP in DMSO-d6 (5 mg in 0.5 mL of DMSO-d6) with addition of DCl (3 µL of 20 wt% in D₂O) indicated two sets of proton signals. One set was assigned to the SP isomer, where eleven aromatic protons were identified explicitly, including the characteristic benzopyran proton (H11) at δ 6.04 ppm. Four methyl groups appeared at δ 2.74, 2.34, 1.28, and 1.22 (Fig. S5). The other set was attributed to the single MC isomer. ¹³C NMR analysis (Fig. S7) also revealed the formation of a single MC isomer. According to a review by W. R. Browne et al.,¹⁹ there are eight possible MC conformers resulting from Z/E configurations around the benzopyran-benzoindoline linkage, as well as potential quinoidal forms. Under normal conditions, the trans-TTC or TTT isomers are predominant. Therefore, we proposed the observed MC species were the TTC isomer (Fig. 2A) due to the following three reasons: (1) rapid conversion of the initially formed cis-CCC isomer (from C_spiro–O cleavage) to the trans-TTC form due to steric strain; (2) the formed MC isomers are zwitterionic, the N in benzoindoline ring is positive while the O in phenoxide ring is negative. The electrostatic interaction prohibits the TTC → TTT conversion. And (3) the characteristic C signal at 184 ppm (Fig. S7), assigned to C2 (C=N⁺), which is distinct from quinoidal carbonyl signals (ca. 195 ppm).

The sensing behavior was investigated by adding various metal ions (Pb²⁺, Zn²⁺, Co²⁺, Ag⁺, Fe^2+/3+, Hg²⁺, Na⁺, Ca²⁺, Mg²⁺, Ni²⁺ and Cu²⁺) to the SP solution. Detailed procedures were provided in the experimental section of the SI. In all tests, the concentration of SP maintained constant at 0.1 mM. With the exception of Cu²⁺, no significant color change was observed upon addition of other metal ions (0.001 mM) to the SP solution (Fig. 3A). Notably, the introduction of Cu²⁺ ions resulted in a distinct color change from colorless to wine-red, readily visible to the naked eye. The color of the solution remains basically unchanged for more than a month, indicating that the formed complex is very stable. UV-vis spectra of the SP solution with various metal cations (Fig. 3B) showed negligible changes in absorption curves except for Cu²⁺, suggesting that SP neither forms stable complexes with other metal ions nor undergoes significant structural alteration. Upon adding Cu²⁺ ions, a new absorption peak at 536 nm increased significantly, indicating a transition from the ring-closed to the ring-open form upon coordination with Cu²⁺.

Fig. 3 (A) Photographs and (B) UV-vis spectra of SP (0.1 mM) with various metal ions (0.01 equiv., 0.001 mM) in CH₃OH/H₂O (1 : 1, v/v). (C) HRMS of SP upon addition of 0.5 equiv. Cu²⁺ in methanol. Structural representations of two SP–Cu²⁺ coordination complexes: the ring-opened merocyanine–Cu²⁺ chelate (complex 1) and the carboxylate–Cu²⁺ complex (complex 2). (D) ¹H NMR titration of SP in DMSO-d6 with incremental Cu(NO₃)₂ equivalents in D₂O at 25 °C. From bottom to top: 0, 0.125, 0.25, 0.5 and 1 equiv. of Cu²⁺. (E) FT-IR spectra of cast films of SP with (bottom) and without (top) Cu²⁺ from methanol.

To confirm the structure of the complex of SP with Cu²⁺, we acquired high-resolution mass spectra (HRMS) of SP with Cu²⁺ in MeOH/H₂O (1 : 1, v/v). As shown in Fig. 3C, two prominent peaks were observed at m/z 996.26767 (singly charged) and 483.63761 (doubly charged), confirming the presence of two distinct types of complexes. The former was corresponded to the carboxylate-bridged complex, [(SP-COO⁻)₂Cu²⁺ + H⁺]⁺ (calculated 996.26899), designated as complex 2 (Fig. 3C). The latter was assigned to the ring-opened merocyanine–Cu²⁺ chelate (complex 1, calculated 483.63841).

Fig. 3D showed the ¹H NMR titration of SP in DMSO-d6 with incremental Cu²⁺ equivalents (in D₂O) at room temperature. Upon addition of Cu²⁺, new proton peaks, attributed to ring-opened merocyanine, emerged and intensified proportionally with increasing Cu²⁺ concentration. At 0.5 equivalents, the integration of these new signals was almost equal to that of the original SP protons, indicating approximately equal concentrations of both isomers. Concurrently, paramagnetic line broadening induced by Cu²⁺ resulted in progressive signal attenuation. This paramagnetic effect was even more pronounced in the ¹³C NMR spectra (Fig. S13), where all carbon peaks disappeared completely at 0.5 equivalents Cu²⁺.

Fig. 3E compared the FT-IR spectra of SP with and without Cu²⁺ ions. For pure SP (Fig. 3E, top), the carbonyl stretching of the –COOH group appeared at 1669 cm⁻¹, indicating the formation of dimer through intermolecular hydrogen bonding. Characteristic vibrations included: C=N stretching (oxazole ring) at 1606 cm⁻¹, C–O stretching (oxazole ring) at 1245 cm⁻¹, benzopyran C–O stretching at 1020 cm⁻¹ and C_spiro–O stretching at 943 cm⁻¹.^22,23 Upon adding 0.5 equiv. of Cu²⁺ (Fig. 3E, bottom), significant spectral changes occurred: (1) The C=O stretching shifted to 1708 cm⁻¹, signifying conversion from dimers to monomers; (2) The C=N stretching (oxazole ring) took a red-shift to 1583 cm⁻¹, indicating coordination through the nitrogen atom to Cu²⁺; (3) the oxazole C–O stretching (1245 cm⁻¹) remained unchanged, confirming that the oxazole oxygen did not participate in coordination; (4) the C_spiro–O band (943 cm⁻¹) decreased markedly, indicating cleavage of the C–O bond. Together with ¹H NMR data, these results confirmed the coexistence of two SP–Cu²⁺ complexes: a carboxylate-bridged dimer and a ring-opened chelate.

To further evaluate the sensitivity of SP toward Cu²⁺, SP solutions containing Cu²⁺ at concentrations ranging from 10⁻⁴ to 10⁻⁸ M were tested (Fig. 4A). A distinct color change from colorless to wine-red was observed even at Cu²⁺ concentrations as low as 10⁻⁷ M. Corresponding UV-vis spectra (Fig. 4B) exhibited a pronounced absorption band at 533 nm for the solution with 10⁻⁷ M Cu²⁺, confirming the high sensitivity of the visual detection method.

Fig. 4 (A) Photographs and (B) UV-vis spectra of SP (0.1 mM) with different concentration of Cu²⁺. Concentration series (A, left to right): [Cu²⁺] = 1.0 × 10⁻⁴, 1.0 × 10⁻⁵, 1.0 × 10⁻⁶, 1.0 × 10⁻⁷ and 1.0 × 10⁻⁸ M. (C) SP-coated test strips (20 µM in THF) after immersion in solutions of various metal ions (10⁻⁶ M) and (D) in aqueous Cu²⁺ solutions at different concentrations. Concentration series ((D), left to right): [Cu²⁺] = 0, 1.0 × 10⁻⁸, 1.0 × 10⁻⁷, 1.0 × 10⁻⁶ and 1.0 × 10⁻⁵ M. (E) Photographs of interference detection of the aqueous solution containing 10⁻⁶ M various metal ions (Pb²⁺, Zn²⁺, Co²⁺, Fe³⁺, Hg²⁺, Ag⁺, Na⁺, Ca²⁺, Mg²⁺, Ni²⁺) with (top) and without (bottom) 10⁻⁷ M Cu²⁺ treated with either SP-coated test strips or SP solution (final [SP] = 10⁻⁴ M). (F) Corresponding UV-vis spectra of the same solutions with and without 10⁻⁷ M Cu²⁺ after addition of SP (final [SP] = 10⁻⁴ M).

Test strips were prepared to facilitate SP-based visual detection of Cu²⁺ with enhanced convenience and selectivity, using the following procedure: Filter paper was immersed in a THF solution of SP (20 mM) and air-dried. The resulting colorless test strips were immersed in aqueous solutions containing 0.001 mM various cations. Strips immersed in Cu²⁺ solutions exhibited a distinct color change from colorless to wine-red (Fig. 4C), readily visible to the naked eye, while those exposed to other cations showed minimal color change. Additionally, test strips were immersed in Cu²⁺ solutions across a concentration range (Fig. 4D). A faint red coloration was observed even at Cu²⁺ concentrations as low as 10⁻⁷ M.

To further evaluate interference from competing ions during the Cu²⁺ detection, we prepared solutions containing various metal ions (Pb²⁺, Zn²⁺, Co²⁺, Fe³⁺, Hg²⁺, Ag⁺, Na⁺, Ca²⁺, Mg²⁺, Ni²⁺) with and without 10⁻⁷ M Cu²⁺. The concentration of all non-copper cations was maintained at 10⁻⁶ M. Upon addition of SP (final concentration: 0.1 mM), the solutions containing Cu²⁺ turned wine-red immediately (Fig. 4E), whereas solutions without Cu²⁺ remained colorless. Test strip analysis further confirmed exceptional selectivity toward Cu²⁺, with strong anti-interference capability against other competing ions. The high sensitivity was additionally verified by UV-vis spectra (Fig. 4F).

To examine the actual application, we determined the concentration of copper ions in the nearshore seawater of Jinsha Bay, Zhanjiang with the benzoxazole-spiropyran probe. The calibration curve was illustrated in Fig. S14. The determined absorbance of seawater was ca. 0.162, indicating [Cu²⁺] is ca. 3.3 × 10⁻⁷ M. The slope of calibration curve is the molar absorption coefficient (ε), which is ca. 4.9 × 10⁵ L mol⁻¹ cm⁻¹. Such high ε value accounted for the exceptional sensitivity of Cu²⁺ detection. The qualitative naked-eye detection (Fig. S15) also revealed that the [Cu²⁺] was about in the range of 10⁻⁶–10⁻⁷ M. To verify our method, the standard method, atomic absorption spectroscopy, was compared to detect the same seawater sample. The corresponded calibration curve was showed in Fig. S16. And the absorbance of seawater was 0.227. Thus, the [Cu²⁺] is ca. 0.015 mg L⁻¹, that is 2.3 × 10⁻⁷ M. The results indicated that our method is generally accurate and reliable.

In summary, we have successfully synthesized a benzoxazole-spiropyran probe and demonstrated exceptional selectivity and sensitivity for Cu²⁺ detection. This spiropyran enabled naked-eye identification of Cu²⁺ at concentrations as low as 10⁻⁷ M in both solution and test strip formats, exhibiting a distinct color change to wine-red. The high molar absorption coefficient (ε = 4.9 × 10⁵ L mol⁻¹ cm⁻¹ at λ_max = 535 nm) and strong anti-interference capability promote its convenient utility as a practical, efficient, and reliable sensor for Cu²⁺ detection.

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (21773103 and 21902070).

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). The synthesis of benzoxazole-spiropyran probe (SP), ¹H NMR spectra of the intermediates and the final SP, the experimental methods of photochromic and acid-chromic test, high-resolution mass spectrum (HRMS) of SP with and without Cu²⁺, images of various metal ions (10⁻⁴ and 10⁻⁵ M) with the SP probe, visual detection to Cu²⁺ in the nearshore seawater and comparison with standard method, Atomic Absorption Spectroscopy (AAS) are available of supplementary information. See DOI: https://doi.org/10.1039/d5cc05144d.

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