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Article

Colorimetric and SERS-Based Multimode Detection Platform for Cu(II) Ions Using Peptide–Gold Nanoparticles

by
Panangattukara Prabhakaran Praveen Kumar
KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-or, Seongbuk-gu, Seoul 02841, Republic of Korea
Colorants 2025, 4(4), 29; https://doi.org/10.3390/colorants4040029
Submission received: 13 August 2025 / Revised: 12 September 2025 / Accepted: 21 September 2025 / Published: 24 September 2025
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Abstract

Excessive copper ions in the human body can cause a variety of diseases, such as gastrointestinal disorders, cirrhosis, and Alzheimer’s disease. Techniques like Inductively Coupled Plasma–Mass Spectroscopy and Atomic Absorption Spectroscopy are available for copper detection, but the associated cost issues for sample preparation and labor limit their application for on-site detection. Herein, we are reporting a versatile method for detecting copper ions using a peptide-functionalized gold nanoparticle sensor in combination with various optical spectroscopic techniques. The peptide (CW) exhibited selective sensing ability for Cu(II) with visual colorimetric and optical spectroscopic changes compared to other metal ions tested. CW showed a visual colorimetric response from colorless to light brown color after interaction with Cu(II). Converting CW to a gold nanoparticle appended (CW-AuNPs) nanoplatform enabled a multimodal detection platform for Cu (II), which utilizes colorimetric and optical spectrum changes and surface-enhanced Raman spectroscopy (SERS) to enable highly sensitive sensing of Cu(II), even at extremely low concentrations (76 nms.). CW-AuNPs exhibit a controlled aggregation property in the presence of Cu(II), resulting in the creation of hot spots for SERS-based detection. Moreover, the peptide unit attached to the gold nanoparticles serves both as a binding motif for Cu(II) and as a Raman reporter for Cu(II) sensing. Our comprehensive analysis, including solution-state and dry-mapping Raman spectroscopic studies, demonstrates remarkable picomolar sensitivity of the peptide–gold nanoparticle system for Cu(II) detection. Moreover, we prepared a paper test strip from CW-AuNPs and used it as a visual colorimetric platform for sensitive detection of copper ions.

1. Introduction

Metal ion sensing carries significant implications across diverse fields. While many metal ions contribute to vital biochemical reactions, a few pose hazards and toxicity risks [1,2]. Essential trace elements, such as zinc, copper, and manganese, are crucial for living organisms, yet their concentrations beyond optimal levels can lead to adverse effects [3]. Among these metals, copper finds wide industrial applications, but its environmental accumulation can result in severe environmental and health concerns [4,5]. Consequently, the development of sensitive and selective detection methods for copper becomes imperative to ensure environmental safety and human well-being. Although standard techniques like Atomic Absorption Spectroscopy [6], Inductively Coupled Plasma–Mass Spectrometry [7,8], and voltametric techniques [9] offer favorable detection limits for a broad concentration range, they suffer from drawbacks, such as costly sample preparation, time-consuming procedures, and the need for bulky equipment. These techniques are unsuitable for on-site applications, and specialized methods are required for biological sample evaluation. Progress in science has yielded novel detection platforms, including colorimetric [10,11], fluorometric [12,13], magnetic resonance imaging [14,15], and strip-based techniques [16,17] for metal ion detection.
Gold nanoparticles (AuNPs) have gained significant attention as promising materials in biosensing applications due to their unique optical and electronic properties [18,19]. The utilization of colorimetric assays with AuNPs has become an appealing approach for detecting a wide range of analytes. Their strong surface-plasmon resonance peaks (SPR) enable monitoring of minute molecular-level changes. The selection of AuNPs is justified by their ability to exhibit size-tunable optical properties and undergo surface modifications. Notably, surface functionalization of AuNPs with peptides offers specific recognition and binding sites for target analytes [20,21]. Peptides are highly desirable ligands for biosensing applications due to their exceptional selectivity and stability and multiple binding sites [22,23]. By combining the plasmonic nature of AuNPs with the metal binding properties of peptides, various metal ion sensors can be fabricated [24]. Peptide-capped AuNPs provide tunability for metal ion sensing by facilitating multipoint interactions between peptides and different metal ions. This assay platform offers advantages like sensitivity, rapid in situ metal ion detection, and visible color changes resulting from NP aggregation. The surface functionalities can be adjusted using a range of peptides to induce such aggregation [25,26].
The alteration of the SPR peaks of AuNPs directly determines the associated color change when they bind to metals. This change can be attributed to factors like variations in particle size, shape, surface chemistry, or interparticle distance during aggregate formation [27]. Consequently, these modifications in the SPR peaks play a pivotal role in significantly enhancing the Raman scattering of molecules near the AuNPs or aggregates through surface-enhanced Raman spectroscopy (SERS) [28,29]. SERS-based sensors have been proven to meet the essential requirements for metal ion sensing, including high sensitivity, selectivity, and suitability for on-site applications, and for biomedical applications [30,31,32]. The fundamental principle underlying this technique lies in the observed alterations in the Raman spectrum of the material adsorbed on the SERS active nanomaterial upon binding with the analyte. The distinctive spectral features of the adsorbed material, in combination with the SERS activity, enable the development of sensitive and accurate assays for detecting metal ions [33,34,35].
In this study, we present a peptide–Au-based nanoparticle system for the selective detection of Cu(II) ions in aqueous media. The dipeptide (CW) containing cystine and tryptophan as a backbone without AuNPs showed excellent binding property towards Cu (II) ions. After conjugating CW with AuNPs, we demonstrated a colorimetric and Raman-based detection assay with high sensitivity and reproducibility for Cu(II) detection. While previous studies have explored the use of various nanoparticles for Cu(II) detection, the application of SERS for sensitive and on-site detection is relatively scarce. Our peptide–Au nanoparticle (CW-AuNPs) system provides a versatile platform for Cu(II) ion detection, employing multiple optical spectroscopy techniques and Raman spectroscopy. The nanoparticles were carefully designed to ensure excellent dispersity, indicating no non-specific interactions between the peptide and Au. Upon the addition of Cu(II) ions, we observed selective aggregation of the Au nanoparticles, distinguishing Cu(II) from other metal ions. Our solution-based SERS measurements demonstrated picomolar sensitivity, while dry-state Raman mapping studies showed femtomolar sensitivity.

2. Materials and Methods

2.1. General Techniques

1H, 13C NMR spectra were recorded on a Bruker Avance II 500 MHz NMR (Rheinstetten, Germany) spectrometer at 25 °C. Absorption spectra were recorded on a Shimadzu UV-2600 UV-visible (Kyoto, Japan) spectrophotometer in 3 mL quartz cuvettes with a path length of 1 cm. Emission spectra were recorded on Hitachi F7000 instrument (Tokyo, Japan) with a slit width of 2 nm. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectropolarimeter (Tokyo, Japan). Fluorescence lifetimes were measured using the time-correlated single-photon counting (TCSPC) technique on a Deltaflex modular fluorescence lifetime system from HORIBA Scientific (Kyoto, Japan) using a nano-LED pulse diode light source. The instrument response function (IRF) of the setup was 200 ps and measured using 1% ludox (colloidal silica) solution. For dynamic light scattering analysis, a Malvern Zetasizer 2000 DLS spectrometer (Malvern, UK) equipped with a 633 nm CW laser was employed. Transmission electron microscopic images were obtained using a Hitachi H-7100 series TEM instrument (Tokyo, Japan). Raman spectral data were acquired using an inverted Raman microscope (NOST, Seoul, Republic of Korea) equipped with a 60× objective lens (0.6 numerical aperture, Olympus, Tokyo, Japan). A 633 nm laser (CNI Laser, Changchun, China) was employed to excite the sample solution. The resulting scattered Raman signals were collected through a 100 µm confocal motorized pinhole and directed to a spectrometer (FEX-MD, NOST, Republic of Korea) fitted with a 1200 g mm−1 grating. The final signal detection was carried out using a charge-modified device camera (Andor DV401A-BVF, Belfast, Northern Ireland).

2.2. Materials

Metal perchlorates, HEPES buffer, HAuCl4, and NaBH4, were purchased from Sigma-Aldrich (Gangnam-gu, Seoul, Republic of Korea).

2.3. Synthesis of CW

CW was prepared based on previous literature reports [32,36]. CW was characterized using 1HNMR, 13CNMR, and through mass spectrometry. 1HNMR: (500 MHz, D2O) ppm δ: 3.06 (dd, J = 15.0, 7.5 Hz, 2H), 3.17 (m, 4H), 3.24 (m, 2H), 3.63 (s, 6H), 4.47 (m, 2H), 4.71 (br s, 2H), 7.09 (s, 2H), 7.18 (s, 4H), 7.45 (br d, 2H), 7.52 (br d, 2H). 13CNMR (125 MHz, D2O): 26.48, 37.83, 51.49, 52.96, 53.94, 108.68, 111.94, 118.21, 119.35, 121.98, 124.48, 126.75, 136.10, 167.66, 173.19. HRMS calcd. for C30H37N6O6S2, m/z = 641.2211, obtained m/z = 641.2241

2.4. Metal Ion Binding Studies Using UV-Visible and Emission Spectroscopy

Solutions of CW were prepared in HEPES buffer (pH = 7.4) at a concentration of 10 μM. Similarly, metal perchlorate solutions (60 μM) were also prepared in HEPES buffer. These metal ions were gradually added to the solutions of CW and for titration. UV-Vis absorption spectra were recorded throughout the process using a Shimadzu UV-2600 double-beam (Kyoto, Japan) spectrophotometer. Titration was carried out until no further spectral changes were observed, indicating saturation. A similar experiment was performed for emission spectroscopy studies, where the samples were excited at 290 nm and the spectrum was recorded using a Hitachi F7000 instrument (Tokyo, Japan) with a slit width of 2 nm.

2.5. Determination of Stoichiometry of Complex

An equimolar solution of CW and metal perchlorate was prepared in HEPES buffer (10 μM). The mole fraction of CW was systematically adjusted from 0.1 to 1. After applying the necessary correction factors, the corresponding changes in absorbance were recorded and plotted as a function of the receptor mole fraction.

2.6. Estimation of Binding Constant via Benesi-Hildebrand Approach

The binding affinity between CW and Cu(II) was evaluated using UV-visible spectroscopy, applying the Benesi–Hildebrand equation. This method enables the calculation of the association constant (K) by analyzing changes in absorbance. The relationship used is
1/A0 − A = 1/(A0 − A∞)K[G] + 1/(A0 − A∞)
In this equation, A and A0 are the absorbance values of CW in the presence and absence of Cu(II), respectively, while A∞ denotes the absorbance at saturation (infinite dilution with Cu(II)).

2.7. Calculation of Limit of Detection (LOD)

The limit of detection (LOD) for dopamine using compound 4 was determined based on the standard method:
LOD = K ∗ Sb/k.
here, K is a constant (typically 3), Sb represents the standard deviation of the blank (ligand-only) solution, and k is the slope derived from the calibration curve. This calculation provides a measure of the sensor’s sensitivity to low concentrations of Cu(II).

2.8. Preparation of CW-AuNPs

An aqueous solution of CW (3 mL, 0.5 mM) was subjected to reduction using NaBH4 (0.2 mL, 10 mM) in the presence of HAuCl4 (1.8 mL, 0.5 mM). This reaction resulted in the formation of CW-AuNPs, which was confirmed by the visual response of the reaction from colorless to a light pink color.

2.9. SERS-Based Detection

For the surface-enhanced Raman scattering (SERS)-based detection of Cu(II), CW-AuNPs (50 μg) were incubated with varying concentrations of Cu(II), ranging from 10−6 M to 10−15 M. These samples were then drop-cast onto glass slides using a silicone isolator. Raman spectra were recorded using a 633 nm laser (6.8 mW, 10 s exposure time) and a 60× objective lens. Following drying, the solid residues containing CW-AuNPs and bound Cu (II) were subjected to Raman mapping. A region approximately 1.5–2.0 mm in diameter on each glass slide was scanned at 25 × 25 pixels resolution, with 10 s of exposure per pixel. Raman mapping images were generated based on the intensity at 1414 cm−1.

2.10. Test Paper Preparation and Optimization Conditions for Sensors

The test paper was developed as follows. Whatman filter paper No. 1 was immersed in a solution of CW (3 mL, 0.5 mM), incubated for an hour at room temperature, and dried at room temperature for an hour. Afterwards, a solution of HAuCl4 (1.8 mL, 0.5 M) was added to the test paper and left for an hour for adsorption in a Petri dish. Following this period, the test paper was coated with CW and Au dipped in a solution of NaBH4 (10 mM). An immediate visible color change was observed on the filter paper from white to light pink. This test paper dried at room temperature overnight. Before using them as sensor strips, the test paper was washed with water to see whether the CW-AuNPs were leaching from the test paper. After confirming the adsorption of CW-AuNPs on the test paper, 20 μL of Cu(II) solution with varying concentrations was added to the test paper. The color started developing after 3–4 min, and the color difference was monitored using a blank background area where no Cu(II) solution added. To check the stability, the test paper was stored at 4 °C, and the analysis was carried out on different days, say 7, 20, and 30 days. To check the sensitivity, solutions of Cu(II) from 0.0–1 μM to 20 μM were added, and the color change was monitored. The effect of temperature on test paper was also tested by keeping them in an oven at varying temperatures, such as 60 and 70 °C. There was a color change for the test paper from pink to blue and violet without Cu(II) addition, indicating that at these temperatures there is an aggregation of NPs in the test paper. The test papers cannot be reused after Cu(II) sensing.

3. Results

3.1. Characterization and Photophysics for CW

CW was synthesized following previous literature reports and characterized by 1H, 13C-NMR, and mass spectrometry techniques (Scheme S1, Figures S1–S3). CW was fully soluble in water and in HEPES buffer due to the presence of free amino groups in the backbone. CW exhibited an absorption peak at 290 nm, corresponding to the tryptophan unit (Figure S4A). Additionally, excitation at 290 nm resulted in a monomer emission band at 330 nm corresponding to tryptophan fluorescence (Figure S4B). CD spectral studies indicated a random coil structure for CW, with a positive band at 220 nm and a negative band at 197 nm (Figure S4C). The design strategy for CW relies on the metal ion binding property of the disulfide bond, the free amine group, and the ability of the indole unit to form cation–pi interactions. Furthermore, the fluorescence properties of tryptophan make it suitable as a signaling unit after the binding event. The disulfide bond can be easily cleaved using reducing agents, enabling the thiolate peptide to be anchored to AuNPs for sensing applications by taking advantage of the surface plasmon properties of AuNPs for sensing applications.

3.2. Metal Ion Detection Using Spectroscopic Techniques

Following the characterization of CW, we proceeded to examine the optical spectroscopic response of CW when exposed to various metal ions in HEPES buffer (10 mM, pH 7.5). Among the various tested metal ions (Na(I), K(I), Cs(I), Mg(II), Ba(II), Ag(I), Cd(II), Zn(II), Pb(II), Mn(II), Cu(II), Hg(II), Fe(II), Co(II), Ni(II), Cr(II), Cr(III), Fe(III)), Cu(II) was found to induce changes in the absorption spectra, as shown in Figure S5A. The addition of Cu(II) resulted in a red shift in the absorption spectrum, accompanied by the emergence of new absorption bands at 460 nm (Figure 1A). These higher-wavelength absorption bands indicated an interaction between Cu(II) and CW, resulting in metal-induced aggregation of CW. This aggregation resulted in a slight color change from colorless to light brown for the Cu(II)-CW aggregates (Figure 1B ). To confirm peptide aggregation, scanning electron microscopy studies were conducted, revealing changes in the morphological features of CW (Figure 1C,D). CW showed vesicle-like morphology with an average diameter of vesicles of 220.9 ± 11.5 nm (Figure 1B). It is widely acknowledged that peptides containing tryptophan can form vesicles due to the pi–pi interaction of indole rings [32,37]. However, the interaction of Cu(II) disrupted this pi stacking due to the strong binding of Cu(II) with the indole rings, which resulted in aggregation of CW (Figure 1D) [23,32]. Consequently, the peptide aggregation caused discernible chiroptical changes in the CD spectrum of CW, characterized by an inversion of the CD signal by the addition of Cu(II) (Figure 1E). This chiroptical change is due to the structural rearrangement and conformational changes in CW because of Cu(II) binding. There are studies that showed that copper binding to amyloid-β peptides can induce β-sheet formation with chiroptical changes in the spectroscopic signals; in a similar way, CW showed a chiral optical inversion after Cu(II) binding [38,39].
Emission spectroscopy studies provided further insights into Cu(II) sensing. Among the various tested metal ions, the introduction of Cu(II) resulted in a reduction of fluorescence intensity at 330 nm after the Cu(II) interaction (Figure 2A and Figure S5B). Notably, the addition of Hg(II) also led to fluorescence quenching, suggesting their ability to bind with CW. To confirm the binding properties in detail, we performed time-resolved spectroscopy experiments for CW with Cu(II) and Hg(II). The quenching for fluorescence can be due to two types of mechanisms; one is static quenching, and the other is dynamic quenching. In static quenching, there will be a ground state complex formation between the fluorophore and metal ions, resulting in a non-fluorescent ground state complex. Meanwhile, dynamic quenching results in random collision between the fluorophore at the excited state and the metal ions, resulting in non-radiative decay, which lowers the fluorescence. To confirm this, time-resolved spectroscopy is an ideal tool where the lifetime of the fluorophore remains the same after complex formation in the ground state in the static type, whereas a decrease in lifetime indicates dynamic-type quenching. Time-resolved studies showed that there is no change in lifetime for tryptophan fluorescence with Cu(II) concentration, indicating a static-type quenching mechanism with strong ground state complex formation. Meanwhile, the fluorescence lifetime decreased with the addition of Hg (II), indicating dynamic-type quenching and no complex formation between CW and Hg (II) (Figure 2B, Table S1) [23,32]. Thus, the observed change in fluorescence with Hg(II) is due to the paramagnetic properties of Hg(II).
The response of CW to Cu(II) exhibited linearity within the concentration range of 0 to 6.0 × 10−7 M, with a detection limit of 0.3 μM (Figure S6A). The association constant for CW with Cu(II) was calculated using the BindFit model for supramolecular interactions [40,41]. The supramolecular BindFit model showed accurate fitting of data for a 1:1 stoichiometry with an association constant of 4.91 × 104 M−1, which was further supported by the Benesi–Hildebrand association constant values (4.86 × 104 M−1) (Figure S6B,C). The stoichiometry of the CW-Cu(II) complex was confirmed further through a Jobs plot analysis, and it showed 1:1 stoichiometry with an inflation point at the 0.5 mole fraction value (Figure S6D). The complexation was further confirmed using ESI mass spectrometry, where the peak at 704.1528 indicated the complex peak for [CW-Cu-H]+ (Figure S7).
The binding mode of Cu(II) with CW was investigated through 1HNMR studies in D2O (Figure 2C). CW showed distinctive NMR peaks for the -S-CH2 proton at 3.26 and 2.98 ppm. Addition of 0.5 equivalent of Cu(II) resulted in a down-field shift of the –S-CH2- protons by 0.3 ppm, indicating a strong interaction between Cu(II) and the –S atom in cystine moiety. Further addition of Cu(II) resulted in an increased multiplicity of the –S-CH2- protons with broadening. The aromatic protons in the tryptophan moiety showed a considerable up-field shift through the addition of two equivalents of Cu(II). It is well-known that tryptophan can induce cation–pi interactions, and this interaction induced an up-field shift of the aromatic protons due to the developed ring current in the tryptophan moiety after Cu(II) binding [23].
To validate complex formation, we have performed density functional theory (DFT) calculations for CW and the CW-Cu(II) complex (Figure 2D–E). The DFT studies were performed using Gaussian 09, implying a B3LYP functional. C, N, O, S, and H atoms were treated with the 6–31G(d) basis set, while Cu was treated with the Lanl2dz basis set. As shown in Figure 2E, Cu(II) (green color) possesses a distorted square pyramidal structure, whereas Cu (II) ions were coordinated with one of the sulphur atoms from cystine (yellow color), indole carbon atoms (grey color), the indole nitrogen atom (blue color), and carbonyl oxygen atoms (red color) (Figure 2E). Cu(II) formed a three-membered ring structure with the indole ring from tryptophan, and this indicates a delocalized pi electron cloud from tryptophan for the stabilization from previous reports. CW showed an average -S-S- bond distance of 2.087 Å with a dihedral angle for -C-S-S- of 103.05° and 105.08°. Meanwhile, in the complex, the average -S-S- bond distance increased slightly, indicating the coordination of Cu(II) to the sulphur atom, which reduces the bond strength in -S-S-. The dihedral angle for -C-S-S- is increased slightly after complex formation (104.04° and 109.21°). The average distance between the benzene rings decreased considerably in the CW-Cu(II) complex (4.983 Å) compared with CW alone (5.599 Å), indicating that Cu(II) binding brings the benzene rings in proximity for stabilizing the complex.
Thus, from the DFT studies, the complexity in 1HNMR spectra after Cu(II) binding can be explained as follows. The observed complex multiplicity in NMR spectra for the S-CH2 protons after the addition of Cu(II) is due to the presence of complexed and free -S-CH2 protons, which resonate at two different chemical shifts. From DFT studies, it is clear that after Cu(II) binding, the distance between benzene rings is reduced; simultaneously, a tricyclic metallic ring is formed with the indole ring and Cu(II). This structure induces a ring current, resulting in the up-field shift of indole aromatic protons, as observed in previous studies [23]. NMR spectra showed there was no change in the chemical shift for COOMe protons, indicating that this carbonyl group is not coordinated with Cu(II), as supported by DFT studies.

3.3. Surface Plasmon Resonance-Based Detection

In recent years, there has been significant advancement in the field of sensing applications with the development of nanoparticles. These nanoparticles have gained attention due to their surface plasmon resonance peaks (SPR), which exhibit a strong and easily detectable color change with analytes. This unique characteristic allows for a straightforward and practical nanoparticle-based detection technique. Among various nanoparticles, gold nanoparticles (AuNPs) show potential sensing applications, as they offer advantages, such as affordability, a wide range of color tunability, and easy surface modifications for sensing applications. The colorimetric response observed in nanoparticle-based detection techniques is attributed to two main factors. Firstly, the color change occurs because of the direct interaction between AuNPs and the analyte being detected [18]. Alternatively, the analyte may interact with surface-capped functionalities on the AuNPs, leading to the aggregation of nanoparticles [42]. To enhance stability and introduce desired functionalities, ligands are often added to the surface of AuNPs, such as amines, thiols, hydroxyl, etc. This led us to investigate the effect of the disulfide bond (-S-S-) in CW, which can be easily reduced to a thiol -SH bond and can attach to AuNPs [43]. This modification not only imparts stability but also enhances the detection sensitivity for metal ions through the strong coupling effect of SPR bands from Au and the absorption features of CW. Consequently, the colorimetric response of AuNPs can be effectively utilized for the detection of Cu(II).

3.3.1. Synthesis of CW-AuNPs and Photophysics

An aqueous solution of CW (3 mL, 0.5 mM) was subjected to reduction using NaBH4 (0.2 mL, 10 mM) in the presence of HAuCl4 (1.8 mL, 0.5 mM). This reaction resulted in the formation of CW-AuNPs, which was confirmed by the visual response of the reaction from colorless to a light pink color (Figure 3A inset). The UV-visible spectrum exhibited an SPR band at 550 nm, indicating the presence of AuNPs (Figure 3A). To determine the size of the AuNPs, transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments were performed. CW-AuNPs showed a hydrodynamic diameter of 105.63 nm with a polydispersity index of 0.082, indicating highly disperse and stable particles (Figure 3B). TEM analysis revealed an average size of 45.1 nm for CW-AuNPs (Figure 3B inset). Fluorescence spectra were recorded for CW-AuNPs, and excitation of CW-AuNPs at 290 nm showed emission spectra with a maximum at 405 nm (Figure 3C). Compared with the emission spectra of CW, the CW-ANPs showed an emission peak at 405 nm, indicating strong aromatic π–π stacking interactions on the metal surface, resulting in an excimer emission peak rather than monomer emission compared to CW [44].
To investigate the capping of CW onto the surface of the AuNPs, Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy were utilized. FTIR studies showed the characteristic peptide peaks on the surface of AuNPs (Figure S8). IR peaks at 1639 and 1522 cm−1 correspond to the amide I and amide II bending vibrations from the CW functional group. Similar IR peaks were observed in the CW-AuNPs with slight shift in the peak positions, such as 1630 for amide I stretch and 1528 for amide II bending, indicating successive capping of CW on AuNPs. Raman spectroscopy was used to provide additional evidence for the capping of CW on the AuNPs (Figure 3D). The characteristic Raman peak at 497 cm−1 corresponding to the disulfide bond was absent in the CW-AuNPs spectrum, indicating the formation of a -S-Au bond. The peaks observed at 1010 indole ring breathing vibration, 1270 and 1326, correspond to the -CH rocking vibrations in the Raman spectrum, which were attributed to the Raman peaks of tryptophan, further confirming the successful capping of CW on the surface of AuNPs [45]. The slight shifts in peak positions of CW-AuNPs compared with CW indicate the interaction of CW with AuNPs.

3.3.2. Metal Ion Sensing for CW-AuNPs

Subsequently, our research focused on exploring the potential of CW-AuNPs as a means of detecting metals in HEPES buffer solution (10 mM, pH 7.5). Among the various metal ions tested, the introduction of Cu(II) to CW-AuNPs triggered a noticeable color change from pink to bluish due to the plasmonic properties of Au (Figure 4A). The addition of Cu(II) resulted in dramatic changes in the absorption spectrum for CW-AuNPs, with the formation of absorption bands at higher wavelengths (Figure 4B). The formation of a higher-wavelength absorption band indicated the aggregation of AuNPs based on the observed color change from pink to blue. There were no changes in the color and absorption spectrum features of CW-AuNPs due to the addition of other metal ions, indicating the selectivity of CW-AuNPs for Cu(II) detection because of the peptide binding moieties on the surface of AuNPs. CW-AuNPs showed a linear relationship with the absorbance at 550 nm at various concentrations of Cu(II), with a limit of detection of 0.076 μM for Cu(II) (Figure 4C). A visual colorimetric response was observed depending on the concentration of Cu(II) added to CW-AuNPs due to the strong coupling of the SPR properties of CW-AuNPs with metal ions (Figure 4C inset).
As another detection mode, next, we utilized the fluorescence from CW-AuNPs as a signal reader for copper detection. The excimer peak at 410 nm corresponding to the tryptophan excimer quenched after Cu(II) addition indicated a strong interaction between CW-AuNPs and Cu(II) (Figure 4D). By incorporating CW into AuNPs, we achieved enhanced sensitivity for copper detection compared with CW alone. The selectivity of CW-AuNPs with various metal ions was further validated from the colorimetric, UV, and emission-based studies, but CW-AuNPs showed selectivity towards Cu(II) (Figure S9A,B). Through competitive binding experiments with other metal ions, we demonstrated that CW-AuNPs can selectively detect Cu(II), even in the presence of other metal ions (Figure S9A,B). TEM studies showed that the addition of Cu(II) induced the aggregation of NPs, which supports the formation of higher-wavelength absorption bands and the observed colorimetric response with Cu(II) (Figure 4E). As Hg(II) can form an amalgam with AuNPs, an observable color change and optical spectrum response was observed with Hg (II) addition, but the changes were visible with a higher concentration of Hg(II) (>50 μM) compared with Cu(II). This indicated the selectivity of CW peptide on the surface of AuNPs for sensing Cu(II) compared to other metal ions (Figure S10).

3.3.3. Paper-Strip-Based Colorimetric Response for Cu(II)

Paper-strip-based sensing platforms offer a low-cost, portable, and user-friendly approach for detecting metal ions, making them ideal for on-site and point-of-care applications. These strips require minimal sample volume and enable rapid visual readouts through color changes, eliminating the need for complex instrumentation. Their biodegradable nature ensures environmental sustainability, while the surface can be easily functionalized for selective and sensitive detection. Additionally, they demonstrate good shelf life and can be engineered for multiplexed sensing, further enhancing their practicality in diverse analytical settings.
For practical application, we developed test strips coated with CW-AuNPs (see Section 2.10). The test strips were air-dried at room temperature under vacuum overnight. The strips were stored at 4 °C for a period of 1 month to assess their shelf stability. The findings revealed that the sensor system maintained excellent stability throughout storage, confirming its potential applicability for reliable detection of Cu(II). Upon treatment with Cu(II), the pads exhibited a light bluish color compared to the untreated (blank) strips, as illustrated in Figure 5A. These visually responsive test strips present a user-friendly, cost-effective, and environmentally sustainable approach for Cu(II) detection. To assess storage stability, the strips were kept for a duration of one month, during which they retained their functionality, indicating strong shelf life and reliability for detecting Cu(II). The test strips demonstrated high selectivity towards Cu(II) with a distinct color transition from pink to blue and eventually to dark blue with increasing concentrations of Cu(II) (Figure 5B).

3.4. Surface-Enhanched Raman-Spectroscopy-Based Detection

Controlled manipulation of nanoparticle aggregation can significantly amplify the formation of localized electromagnetic fields, commonly known as “hot spots,” which are highly advantageous for SERS applications. Figure 6A illustrates the underlying concept of using SERS nanoprobe technology to detect Cu(II). This detection relies on the unique interaction between CW and Cu(II), resulting in alterations in the SERS intensity of CW appended to AuNPs. These intensity changes are directly proportional to the concentration of Cu(II). Cu(II) can strongly bind with the free amino groups and the indole moiety of CW and result in the aggregation of CW-AuNPs. As shown in Figure 3D, the CW powder sample barely showed any vibrational characteristics in the Raman spectrum. But, incorporation of CW with AuNPs showed vibrational peaks with an enhanced Raman signal intensity for the characteristic functional moieties in CW. In this context, when CW-AuNPs are exposed to Cu(II) ions, they exhibit an aggregation phenomenon; by leveraging this aggregation behavior, the detection of Cu(II) can be achieved using a more sensitive SERS technique. The addition of Cu(II) induces changes in the characteristic Raman peaks of CW-AuNPs, and these changes exhibit a linear correlation with the concentration of Cu(II). Raman analysis was performed on both the solution and solid states (Raman mapping) in the presence of Cu(II). The aggregation of CW-AuNPs with Cu(II) leads to an enhancement in the Raman intensity, thus improving the SERS signal of the system. Here, CW acts as a Raman reporter and showed enhanced SERS due to the aggregation of AuNPs by Cu (II) to form the so-called hot spots.
As shown in Figure 6B, the addition of Cu(II) increased the SERS intensity of CW-AuNPs. To optimize the sensitivity of copper ion detection, we relied on the SERS peak at 1416 cm−1 because of its high intensity after copper-induced aggregation. According to the strategy of Cu(II) detection, the concentration of Cu(II) plays an essential role in the aggregation of CW-AuNPs and in the formation of hot spots for SERS-based detection. Thus, Raman spectral changes of CW-AuNPs were monitored using varying concentrations of Cu(II) from 1 × 10−5 M to 1 × 10−15 M. The dose-dependent curve showed a linear correlation for the most intense Raman peak in CW with varying concentrations of Cu(II) (Figure 6C). It was observed that CW-AuNPs showed a detectable Raman intensity for the peak even at a lower concentration of Cu(II), such as 10 nM, indicating a highly sensitive detection platform compared to optical spectroscopy-based detection. We have studied selectivity in Cu(II) detection using a SERS study and other potential competitive metal ions. Interestingly, SERS studies showed Raman signal enhancement for the peak at 1416 cm−1 for Cu(II) even in the presence of other metal ions tested (Figure 6D). Raman mapping studies were performed for CW-AuNPs with varying concentrations of Cu(II) (Figure 6E). Because Raman mapping is performed with dry samples, it offers enhanced aggregation and, in turn, hot spot formation for SERS applications. The mapping images showed a detectable signal pattern with bright spots for a lower concentration of Cu(II) up to 10 pM (Figure 6F). The bright red spots indicate a measure of the Raman spectrum intensity for the peak at 1416 cm−1 (Figure 6F). Thus, both solution state and dry Raman mapping studies improved the detection limit for Cu(II) using CW-AuNPs compared to optical spectroscopic-based detection methods.

4. Discussion

The present work introduces a peptide-functionalized gold nanoparticle (CW-AuNPs) platform that achieves ultra-sensitive and selective detection of Cu(II) ions through a synergistic combination of peptide recognition and plasmonic signal amplification. The CW peptide, rich in tryptophan and cystine residues, facilitates strong coordination with Cu(II), triggering nanoparticle aggregation and inducing distinct optical changes. This aggregation not only alters the surface plasmon resonance band but is also detectable using colorimetric and UV-visible spectroscopy measurements and generates nanoscale “hot spots” that dramatically enhance Raman scattering signals, enabling detection down to 10 pM in SERS-based assays. Such detection limits surpass most reported peptide nanoparticle sensors, highlighting the exceptional signal enhancement achievable via controlled nanostructure assembly. Importantly, the CW-AuNPs retained high specificity in the presence of competing biologically relevant metal ions, which is critical for deployment in complex biological and environmental samples. The development of a solid-phase sensing format, in the form of functionalized paper strips, further underscores the practicality of the platform for rapid, on-site Cu(II) monitoring without the need for sophisticated instrumentation. A comparative analysis with previously reported Cu(II) sensors (Table 1) highlights the superior sensitivity of the CW-AuNPs platform, with detection limits several orders of magnitude lower than most nanoparticle- or peptide-based probes, alongside excellent selectivity.

5. Conclusions

In summary, we have successfully demonstrated a peptide–gold nanoparticle system with multimode detection capability for Cu(II) metal ions. The prepared CW-AuNPs leverage the advantage of peptide moiety for selective and sensitive binding of Cu(II) among all other metal ions tested. The NPs showed colorimetric, optical, and SERS-based sensitive detection for Cu(II). Optical-based detection allowed for sensitivity up to 76 nM, whereas the SERS-based study offered 10 pM, with high reproducibility for both solution and mapping studies. A paper strip developed from these NPs allowed for on-site application for the detection of Cu(II). Overall, our results highlight the potential of peptide–AuNP conjugates as a versatile and robust platform for copper ion detection, addressing current challenges in sensitivity, selectivity, and cost-effectiveness. Future studies could extend this strategy to multiplex sensing or real-sample testing in complex matrices, such as serum, wastewater, and food extracts.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/colorants4040029/s1, Scheme S1. Synthetic route for the peptide sensor CW. Figure S1. 1H NMR spectrum (D2O, 300 MHz) of CW; Figure S2. 13C NMR spectrum (D2O, 75 MHz) of CW; Figure S3. Mass spectrometry data for CW. Figure S4. Photophysics for CW; Figure S5. Metal ion selectivity study for CW with various metal ions; Table S1: Time-resolved spectroscopic data; Figure S6. Binding constant and stoichiometry measurements. Figure S7. ESI-MS for CW-Cu complex. Figure S8. FT-IR data for CW-AuNPs; Figure S9. Metal ion selectivity study for CW-AuNPs with various metal ions. Figure S10. UV-visible spectra for CW-AuNPs with Hg (II).

Funding

KU-KIST Graduate School of Converging Science and Technology, BK21 fellowship program, Korea University. Science and Engineering Research Board (SERB), New Delhi, India (PDF/2017/001221).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Acknowledgments

P.P.P.K. expresses sincere gratitude to Dong-Kwon Lim, KUKIST, South Korea and BK21 fellowship program, South Korea. P.P.P.K. acknowledges Prakash P. Neelakandan, INST, Mohali, Punjab, India, for accepting the short-term visit to his lab and the help provided by him for the synthesis of the CW peptide. The author acknowledges the instrumentation facilities at KUKIST and INST Mohali for this work.

Conflicts of Interest

The author declares no conflicts of interest.

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Figure 1. Binding studies for CW with Cu(II). (A) Changes in the absorption spectra for CW (10 μM) alone and with various amounts of Cu(ClO4)2·6H2O in HEPES buffer (pH =7.4). The red dashed arrows indicate the changes in absorption spectra for CW with various amount of Cu(II). (B) Photographs showing the color change of CW with increasing amounts of Cu(II). Scanning electron microscopy images for CW without (C,D) with Cu(ClO4)2·6H2O [60 μM]. Scale bar 1 μm. (E) Circular dichroism spectra for CW (60 μM) alone and with the addition of various amounts of Cu(ClO4)2·6H2O.
Figure 1. Binding studies for CW with Cu(II). (A) Changes in the absorption spectra for CW (10 μM) alone and with various amounts of Cu(ClO4)2·6H2O in HEPES buffer (pH =7.4). The red dashed arrows indicate the changes in absorption spectra for CW with various amount of Cu(II). (B) Photographs showing the color change of CW with increasing amounts of Cu(II). Scanning electron microscopy images for CW without (C,D) with Cu(ClO4)2·6H2O [60 μM]. Scale bar 1 μm. (E) Circular dichroism spectra for CW (60 μM) alone and with the addition of various amounts of Cu(ClO4)2·6H2O.
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Figure 2. Binding studies for CW with Cu(II). (A) Changes in the emission spectra for CW (10 × 10−6 M) alone and with various amount of Cu (ClO4)2·6H2O in HEPES buffer (pH = 7.4). The red dotted line indicates the decrease in fluorescence intensity of CW upon addition of varying amounts of Cu(II). (B) Time-resolved spectra for CW with Cu(II). (C) Partial 1H-NMR spectra for CW, and CW with various amounts of Cu(II). The NMR experiments were performed in D2O. The green dotted lines indicate the changes in NMR peak positions for CW and after the addition of Cu(II). (D) Optimized structure for the CW and (E) optimized structure for CW with Cu(II).
Figure 2. Binding studies for CW with Cu(II). (A) Changes in the emission spectra for CW (10 × 10−6 M) alone and with various amount of Cu (ClO4)2·6H2O in HEPES buffer (pH = 7.4). The red dotted line indicates the decrease in fluorescence intensity of CW upon addition of varying amounts of Cu(II). (B) Time-resolved spectra for CW with Cu(II). (C) Partial 1H-NMR spectra for CW, and CW with various amounts of Cu(II). The NMR experiments were performed in D2O. The green dotted lines indicate the changes in NMR peak positions for CW and after the addition of Cu(II). (D) Optimized structure for the CW and (E) optimized structure for CW with Cu(II).
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Figure 3. Characterization of CW-AuNPs. (A) Extinction spectra for CW-AuNPs (100 μg/mL) in HEPES buffer. (B) Dynamic light scattering measurement for CW-AuNPs. The inset showing transmission electron microscopic image for CW-AuNPs. Scale bar 100 nm. (C) Emission spectra for CW-AuNPs (100 μg/mL) in HEPES buffer. λexc = 290 nm. (D) Raman spectrum for CW and CW-AuNPs. Data were collected using a laser source of excitation at 633 nm laser (6.8 mW); exposure time: 10 s.
Figure 3. Characterization of CW-AuNPs. (A) Extinction spectra for CW-AuNPs (100 μg/mL) in HEPES buffer. (B) Dynamic light scattering measurement for CW-AuNPs. The inset showing transmission electron microscopic image for CW-AuNPs. Scale bar 100 nm. (C) Emission spectra for CW-AuNPs (100 μg/mL) in HEPES buffer. λexc = 290 nm. (D) Raman spectrum for CW and CW-AuNPs. Data were collected using a laser source of excitation at 633 nm laser (6.8 mW); exposure time: 10 s.
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Figure 4. Metal sensing properties of CW-AuNPs. (A) Visual colorimetric response for CW-AuNPs with various metal ions. [CW-AuNPs (100 μg/mL); metal ions 20 μM]. (B) Changes in the extinction spectra for CW-AuNPs with Cu(II), where [CW-AuNPs (100 μg/mL); Cu(II) 20 μM]. The dotted red lines represent the observed changes in the extinction spectra of CW-AuNPs upon the addition of varying amounts of Cu(II). (C) A quantitative measurement for the detection of Cu(II) using CW-AuNPs. Inset showing the visual colorimetric response of CW-AuNPs with various amounts of Cu(II) added. (D) Changes in the emission spectra for CW-AuNPs with Cu(II), where [CW-AuNPs (100 μg/mL); Cu(II) 20 μM]. λexc = 290 nm. The dotted red lines indicate the decrease in emission intensity upon the addition of varying amounts of Cu(II). (E) TEM micrographs for CW-AuNPs with Cu(II). Scale bar 100 nm.
Figure 4. Metal sensing properties of CW-AuNPs. (A) Visual colorimetric response for CW-AuNPs with various metal ions. [CW-AuNPs (100 μg/mL); metal ions 20 μM]. (B) Changes in the extinction spectra for CW-AuNPs with Cu(II), where [CW-AuNPs (100 μg/mL); Cu(II) 20 μM]. The dotted red lines represent the observed changes in the extinction spectra of CW-AuNPs upon the addition of varying amounts of Cu(II). (C) A quantitative measurement for the detection of Cu(II) using CW-AuNPs. Inset showing the visual colorimetric response of CW-AuNPs with various amounts of Cu(II) added. (D) Changes in the emission spectra for CW-AuNPs with Cu(II), where [CW-AuNPs (100 μg/mL); Cu(II) 20 μM]. λexc = 290 nm. The dotted red lines indicate the decrease in emission intensity upon the addition of varying amounts of Cu(II). (E) TEM micrographs for CW-AuNPs with Cu(II). Scale bar 100 nm.
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Figure 5. Paper-strip-based sensing of Cu(II). (A) Test strip prepared from CW-AuNPs. (B) Colorimetric response of paper strip with varying concentration of Cu(II).
Figure 5. Paper-strip-based sensing of Cu(II). (A) Test strip prepared from CW-AuNPs. (B) Colorimetric response of paper strip with varying concentration of Cu(II).
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Figure 6. SERS-based copper detection. (A) A schematic representation of SERS-based Cu(II) detection using CW-AuNPs. (B) Raman spectra for CW-AuNPs (50 μg/mL) with the addition of various amounts of Cu(II). The dotted lines indicate the increase in Raman intensity at 1416 cm−1 upon the addition of increasing amounts of Cu(II). (C) Linear relationship between SERS peak intensities at 1416 cm−1 and Cu(II) concentrations. (D) Raman spectra for CW-AuNPs, CW-AuNPs + Cu(II), and CW-AuNPs + Cu(II) + various other metal ions. Studies showed high selectivity of CW-AuNPs with Cu(II), even with the addition of various other metal ions. (E) SERS for the dried samples of CW-AuNPs with Cu(II). Blue shaded box indicates Raman shift at 1416 cm−1. (F) Raman mapping images for the dried samples from CW-AuNPs with various concentrations of Cu(II). Excitation: 633 nm laser (6.8 mW); exposure time: 10 s.
Figure 6. SERS-based copper detection. (A) A schematic representation of SERS-based Cu(II) detection using CW-AuNPs. (B) Raman spectra for CW-AuNPs (50 μg/mL) with the addition of various amounts of Cu(II). The dotted lines indicate the increase in Raman intensity at 1416 cm−1 upon the addition of increasing amounts of Cu(II). (C) Linear relationship between SERS peak intensities at 1416 cm−1 and Cu(II) concentrations. (D) Raman spectra for CW-AuNPs, CW-AuNPs + Cu(II), and CW-AuNPs + Cu(II) + various other metal ions. Studies showed high selectivity of CW-AuNPs with Cu(II), even with the addition of various other metal ions. (E) SERS for the dried samples of CW-AuNPs with Cu(II). Blue shaded box indicates Raman shift at 1416 cm−1. (F) Raman mapping images for the dried samples from CW-AuNPs with various concentrations of Cu(II). Excitation: 633 nm laser (6.8 mW); exposure time: 10 s.
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Table 1. Comparison of reported Cu(II) detection systems with the present CW-AuNPs platform.
Table 1. Comparison of reported Cu(II) detection systems with the present CW-AuNPs platform.
Sensor TypeMode of DetectionLimit of DetectionRef
Julolidine-containing naphthol-based probeColorimetric2 μM[46]
Gold nanoparticles
(AuNPs)		Colorimetric and UV-visible0.04 μM[47]
Papain-functionalized AuNPsColorimetric 200 nM[48]
Silver-coated gold nanoparticlesColorimetric1 nM[49]
H-CVNITKQHTVTTTT-NH2 (peptide)Electrochemical 80 nM [50]
Peptide–chelatorFluorescence 2 μM[51]
Up-conversion lanthanidesLuminescence37 nmol/L[52]
Molecularly imprinted nanofilmSurface plasmon resonance0.027 µM[53]
Molecularly imprinted nanoparticlesSurface plasmon resonanceNA[54]
Pyridine––AuNPsSERS 50 nM[35]
Silver nanoparticlesSERS10 pM[55]
Peptide–AuNPsSERS, colorimetric, fluorescence, paper strip0.3 μM through optical spectroscopy
76 nM with SPR detection
10 pM through SERS		Present work
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Kumar, P.P.P. Colorimetric and SERS-Based Multimode Detection Platform for Cu(II) Ions Using Peptide–Gold Nanoparticles. Colorants 2025, 4, 29. https://doi.org/10.3390/colorants4040029

AMA Style

Kumar PPP. Colorimetric and SERS-Based Multimode Detection Platform for Cu(II) Ions Using Peptide–Gold Nanoparticles. Colorants. 2025; 4(4):29. https://doi.org/10.3390/colorants4040029

Chicago/Turabian Style

Kumar, Panangattukara Prabhakaran Praveen. 2025. "Colorimetric and SERS-Based Multimode Detection Platform for Cu(II) Ions Using Peptide–Gold Nanoparticles" Colorants 4, no. 4: 29. https://doi.org/10.3390/colorants4040029

APA Style

Kumar, P. P. P. (2025). Colorimetric and SERS-Based Multimode Detection Platform for Cu(II) Ions Using Peptide–Gold Nanoparticles. Colorants, 4(4), 29. https://doi.org/10.3390/colorants4040029

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