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. 2026 Jul 3;16(35):35739–35751. doi: 10.1039/d6ra03509d

Green synthesis of silver and zinc oxide nanoparticles for SERS-based detection of resonant fluorescent molecules

Muhammad Khuzaima Zaka a,†, Aftab Farrukh b,†,✉, Amjad Ali c, Mudassir Hussain Tahir d, Sultan Muhammad a, Jaweria Ambreen a, Syeda Bakhtawar Taimoor a, Naushad Ahmad e
PMCID: PMC13329862  PMID: 42405135

Abstract

Strong fluorescence from resonant organic dyes often limits the applicability of Raman spectroscopy by masking weak vibrational signals. In this study, green-synthesized silver nanowires (Ag NWs) and zinc oxide nanoparticles (ZnO NPs) derived from Azadirachta indica leaf extract were investigated as surface-enhanced Raman scattering (SERS) substrates for the fluorescent dyes resorufin and pyronin-Y. The synthesized nanostructures were characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning and transmission electron microscopy (SEM/TEM), and energy-dispersive spectroscopy (EDS). Ag NWs exhibited a face-centered cubic structure with an interconnected wire-like morphology, whereas ZnO NPs possessed a highly crystalline hexagonal wurtzite structure with a spherical morphology and smaller particle size. The optical properties of resorufin and pyronin-Y were examined through Raman and photoluminescence spectroscopy under 532 nm excitation. Both dyes exhibited intense fluorescence backgrounds that obscured their Raman signatures. The incorporation of Ag NWs and ZnO NPs significantly improved spectral resolution by suppressing fluorescence and enhancing Raman scattering. Distinct vibrational bands associated with aromatic ring deformations and C–C, C–N, C Created by potrace 1.16, written by Peter Selinger 2001-2019 C, C Created by potrace 1.16, written by Peter Selinger 2001-2019 N, and C Created by potrace 1.16, written by Peter Selinger 2001-2019 O vibrations were successfully identified. Among the investigated substrates, ZnO nanoparticles produced markedly stronger Raman enhancement than Ag nanowires, particularly for the highly fluorescent analytes studied. The enhanced SERS activity of ZnO is attributed to the combined effects of efficient light scattering, high surface area, and defect-mediated charge transfer between the semiconductor surface and the adsorbed molecules. The proposed energy-level alignment further supports a chemical enhancement pathway under resonant excitation conditions. This study demonstrates the potential of green-synthesized ZnO nanostructures as sustainable and efficient SERS substrates for fluorescence-suppressed molecular detection and provides new insight into semiconductor-assisted Raman enhancement mechanisms.

Strong fluorescence from resonant organic dyes often limits the applicability of Raman spectroscopy by masking weak vibrational signals.graphic file with name d6ra03509d-ga.jpg

1. Introduction

Surface-Enhanced Raman Spectroscopy (SERS) has found extensive utilization in practical scenarios across a multitude of disciplines, including the detection of analytes, forensic investigations, drug delivery and imaging, ensuring food safety, and identifying environmental pollutants.1,2 The technique significantly amplifies the Raman scattering intensity of molecules that are adsorbed on or in close proximity to SERS-responsive substrates.3,4 This amplification can achieve enhancements of many orders of magnitude, thereby facilitating the detection and analysis of molecules at extremely low concentrations.5,6 Noble metals, especially gold (Au) and silver (Ag), are widely recognized as the most effective substrates for Surface-Enhanced Raman Scattering (SERS) measurements.5,7 However, these advanced substrates are beset by several limitations that have impeded their broader adoption and further development.8,9 Key drawbacks include: low homogeneity,10 rapid oxidation and aggregation11 and limited selection of SERS-active metals.12 These challenges necessitate ongoing research to develop alternative or improved SERS substrates that can overcome these inherent shortcomings while maintaining or enhancing the sensitivity and reliability of SERS techniques.

Localized surface plasmon resonance (LSPR) properties of Ag and Au nanostructures generate strong electromagnetic fields that enhance Raman scattering.13,14 Pyronin-Y and resorufin, xanthene-based cationic and resazurin-derived molecules,15 are vital in fluorescence microscopy, nucleic acid staining, bioimaging, and optical sensing due to their strong fluorescence and photostability.16,17 Their fluorescence often interferes with Raman characterization, posing a challenge for spectroscopic studies.18,19 Recent research has focused on sustainable nanoparticle synthesis using plant extracts as reducing and stabilizing agents,20 offering an economical and environmentally friendly approach.21,22 Comparative studies on green-synthesized Ag and ZnO nanoparticles for SERS detection of fluorescent dyes like pyronin-Y and resorufin remain limited.23

Nanoparticles can be produced via a spectrum of methodologies, including chemical, physical, and biological pathways,24 each tailored to specific conditions for optimal results. The contemporary scientific community has increasingly directed its focus toward green synthesis techniques, primarily due to their environmentally benign, economically viable, and non-toxic attributes.25 These unique characteristics of green synthesis have facilitated a range of promising applications, notably in the realm of green electrochemical sensor development. Metallic nanoparticles, exemplified by silver nanoparticles (Ag NPs), have been extensively employed to prolong the shelf life of fresh produce.26 It has been particularly observed that there is a strong draw toward the synthesis of silver (Ag) and zinc oxide (ZnO) nanoparticles, owing to their pronounced antimicrobial properties.27 Silver nanoparticles have emerged as a central focus for researchers,28 attributed to their robust antiseptic qualities and efficacy as potent antibiotic agents.29 The distinctive antimicrobial attributes of zinc oxide nanoparticles (ZnO NPs) have led to their widespread utilization across various domains,30 especially in enhancing the longevity of fruits.31 Furthermore, it has been observed that ZnO NPs exhibit superior antibacterial efficacy in wastewater treatment processes compared to traditional organic antimicrobial agents.32 Recent progress in nanoscience has empowered researchers to develop high-quality fruits with diminished postharvest losses through innovative applications.8 These advancements highlight the potential of nanoparticle technology in tackling challenges related to agricultural sustainability and food security, underscoring its critical role in contemporary scientific and technological endeavors.

In the present work, the green synthesized Ag and ZnO nanoparticles using the extract of Azadirachta indica leaves were studied as the SERS substrate for the resonant fluorescent molecules. A special focus was the mechanism of pyronin-Y and resorufin fluorescence suppression and Raman enhancement. The synthesized nanostructures were systematically analyzed by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), field-emission scanning electron microscopy (FESEM), and energy-dispersive spectroscopy (EDS). The comparative SERS investigations showed that the Raman enhancement of ZnO nanoparticles was much more significant than that of Ag nanoparticles for both analytes. The superior performance of ZnO is suggested to be due to its porous structure, enhanced scattering capability of photons, and defect-induced charge transfer interactions. The present study offers valuable insights into the mechanisms of semiconductor-induced SERS enhancement and opens the door to green-synthesized ZnO nanoparticles as environment-friendly and cost-effective substitutes for traditional noble-metal SERS substrates for various applications such as biosensing, molecular imaging, environmental monitoring, and advanced optical sensing technologies.

2. Materials and methods

2.1. Materials

Silver nitrate (AgNO3), zinc acetate dihydrate [Zn(CH3CO2)2·2H2O], and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich and used without further purification. Fresh leaves of Azadirachta indica (neem) were collected from the campus of PMAS-Arid Agriculture University, Rawalpindi, Pakistan. Deionized water was used throughout the synthesis and sample preparation processes.

2.2. Preparation of neem leaf extract

Fresh and healthy neem (Azadirachta indica) leaves were collected and thoroughly washed with distilled water to remove dust and other impurities. Approximately 5 g of leaves were cut into small pieces and ground into a fine paste using a mortar and pestle. Subsequently, 100 mL of distilled water was added, and the mixture was stirred at 60 °C for 15–20 min to facilitate the extraction of bioactive compounds. The resulting suspension was filtered to remove solid residues, and the filtrate was collected in a clean glass container. The aqueous leaf extract was stored at 4 °C and used as a reducing and stabilizing agent for the green synthesis of silver and zinc oxide nanoparticles.

2.3. Green synthesis of silver nanoparticles (AgNPs)

Silver nanoparticles (AgNPs) were synthesized using the aqueous neem leaf extract as a reducing agent. A silver nitrate solution was prepared by dissolving AgNO3 in 100 mL of distilled water under continuous magnetic stirring. The neem leaf extract was then added dropwise to the AgNO3 solution while maintaining constant stirring. The formation of AgNPs was confirmed by a visible color change from colorless to yellowish-brown, indicating the reduction of Ag+ ions to metallic silver nanoparticles. This color change is attributed to the surface plasmon resonance (SPR) phenomenon characteristic of AgNPs. A schematic illustration of the synthesis procedure is presented in Fig. 1.

Fig. 1. Schematic flowchart showing the green synthesis method of Ag NWs by using neem leaf extract (Azadirachta indica).

Fig. 1

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2.4. Green synthesis of zinc oxide nanoparticles (ZnO NPs)

Zinc oxide nanoparticles (ZnO NPs) were synthesized using zinc acetate dihydrate as the precursor and neem leaf extract as the reducing and capping agent. Zinc acetate dihydrate was dissolved in 100 mL of distilled water under continuous magnetic stirring to obtain a homogeneous solution. The aqueous neem leaf extract was added dropwise to the precursor solution with constant stirring. Subsequently, NaOH solution was added dropwise to adjust the pH and promote nanoparticle formation. The reaction mixture was continuously stirred for 2 h, during which a dark yellow coloration developed, indicating the formation of ZnO nanoparticles.

The resulting precipitate was collected and calcined in a muffle furnace at 400 °C for 3 h to obtain crystalline ZnO nanopowder. The calcined product was ground into a fine powder and stored in an airtight container for further characterization and application. A schematic representation of the synthesis process is shown in Fig. 2.

Fig. 2. Schematic flowchart showing the green synthesis method of ZnO NPs by utilizing neem leaf extract (Azadirachta indica).

Fig. 2

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2.5. Preparation of SERS substrates and analyte samples

Samples for Raman spectroscopy were prepared using AgNPs and ZnO NPs as SERS-active substrates. Equimolar solutions (0.05 M) of the synthesized nanoparticles and analytes (resorufin and pyronin Y) were prepared separately in ethanol. Each solution was sonicated for 30 min to ensure uniform dispersion and complete homogenization.

Clean glass substrates were coated with the nanoparticle suspensions by drop-casting and allowed to dry under ambient conditions. Subsequently, the analyte solution was drop-cast onto the nanoparticle-coated substrates. The substrates were then subjected to spin coating to achieve uniform analyte distribution and improve adsorption onto the nanoparticle surface. The prepared substrates were used for Raman spectroscopic measurements.

3. Results and discussion

3.1. Structural, morphological, and compositional characterization

The successful green synthesis of Ag nanowires (Ag NWs) and ZnO nanoparticles (ZnO NPs) using Azadirachta indica leaf extract was confirmed through XRD, FTIR, SEM/TEM, and EDS analyses.

XRD analysis revealed that Ag NWs possess a face-centered cubic (FCC) crystal structure, whereas ZnO NPs exhibit the characteristic hexagonal wurtzite phase (see Fig. 3a and b). No significant impurity peaks were observed, indicating high phase purity of both nanomaterials. The average crystallite size of ZnO NPs (38.5 nm) was larger than that of Ag NWs (27.7 nm), while ZnO exhibited lower lattice strain (0.00304) compared with Ag NWs (0.0047), suggesting superior crystallinity and fewer structural defects.

Fig. 3. XRD analysis of (a) Ag NWs and (b) ZnO NPs.

Fig. 3

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Morphological characterization by SEM and TEM (Fig. 4) revealed distinct nanostructures for the two materials. Ag NWs formed interconnected wire-like networks with diameters ranging from 70–100 nm and an average size of 86.11 nm.

Fig. 4. (a) and (b) SEM images and (c) and (d) TEM images of Ag NWs and ZnO NPs.

Fig. 4

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This three-dimensional architecture provides numerous junctions and plasmonic hot spots that are advantageous for SERS enhancement.33 In contrast, ZnO NPs exhibited a predominantly spherical morphology with particle sizes between 20 and 40 nm and an average size of 26.15 nm. The SEM micrographs further revealed a porous and slightly irregular surface morphology, suggesting the presence of structural defects and surface-active sites.34 Such defect-rich porous structures can facilitate charge-transfer interactions between the substrate and adsorbed molecules, thereby contributing to the observed SERS enhancement. The high surface area associated with the porous morphology also provides abundant adsorption sites for analyte molecules,35 further improving Raman signal amplification.

TEM observations were consistent with SEM results, confirming the formation of uniform Ag nanowires and well-dispersed ZnO nanoparticles. The smaller size and higher surface-to-volume ratio of ZnO NPs may favor catalytic and adsorption processes,36 whereas the interconnected Ag NW network is beneficial for electromagnetic field amplification. From the particle size distribution graph (Fig. 5a), the average diameter of the Ag NWs was determined to be 86.11 nm. Similarly, the average particle size of the ZnO NPs was calculated to be 26.15 nm (Fig. 5b).

Fig. 5. Particle distribution curves of (a) Ag NWs and (b) ZnO NPs.

Fig. 5

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The elemental composition determined by EDS analysis (Fig. 6) further confirmed the successful synthesis of both nanomaterials. Ag NWs displayed a dominant Ag signal (87.1 wt%), while ZnO NPs showed strong Zn and O peaks characteristic of ZnO. The EDS analysis revealed minor amounts of aluminum (2.4 wt%), ytterbium (0.8 wt%), and silicon (0.5 wt%) in the samples. These elements are attributed to the substrate, sample holder, instrumental components, or trace environmental contamination during sample preparation and analysis. Given their low concentrations and the absence of any evidence of their incorporation into the Ag nanowire or ZnO nanoparticle crystal structures, their contribution to the charge-transfer process is expected to be negligible. Therefore, the observed SERS enhancement is primarily governed by the intrinsic properties of the Ag and ZnO nanostructures, including localized electromagnetic field enhancement and defect-mediated charge-transfer interactions.37 Minor carbon and oxygen signals detected in both samples are attributed to residual phytochemicals adsorbed on the nanoparticle surfaces, which contribute to nanoparticle stabilization and surface functionalization.38

Fig. 6. EDS analysis of (a) Ag NPs and (b) ZnO NPs.

Fig. 6

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Overall, the combined structural, morphological, and compositional analyses confirm the formation of highly crystalline and surface-functionalized Ag NWs and ZnO NPs. While Ag NWs exhibit an interconnected nanowire network favorable for plasmonic and SERS applications, ZnO NPs possess smaller particle size, higher crystallinity, and spherical morphology, making them attractive for catalytic, sensing, and environmental applications.

3.2. Chemistry of resonant fluorescent molecules

Resorufin (7-hydroxy-3H-phenoxazin-3-one) and pyronin-Y (6-(dimethylamino)-N,N-dimethyl-3H-xanthen-3-iminium) were selected as probe molecules because of their strong visible-light absorption and fluorescence properties (Fig. 7). Both molecules possess highly conjugated π-electron systems, which facilitate efficient electronic transitions and make them suitable candidates for resonance Raman and surface-enhanced Raman scattering (SERS) studies.

Fig. 7. Resonant fluorescent molecules (a) resorufin (7-hydroxy-3H-phenoxazin-3-one) (b) pyronin-Y (6-(dimethylamino)-N,N-dimethyl-3H-xanthen-3-iminium).

Fig. 7

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Resorufin is a phenoxazine-based fluorophore derived from the reduction of resazurin.39 Its extended conjugation and carbonyl functionality contribute to strong absorption in the visible region and intense red fluorescence.40 The molecule exhibits pH-dependent optical behavior due to the presence of hydroxyl and carbonyl groups, which influence its electronic structure. The HOMO and LUMO energy levels of resorufin are reported to be −4.38 and −2.74 eV, respectively, indicating an electronic band gap favorable for resonance excitation.

Pyronin-Y is a cationic xanthene dye consisting of a rigid tricyclic aromatic framework with electron-donating dimethylamino groups. The highly conjugated structure enhances its absorption and fluorescence efficiency in the orange-red spectral region. Owing to its positive charge and delocalized π-electron system, pyronin-Y exhibits strong interactions with metallic nanostructures and biological molecules. The HOMO and LUMO energy levels of pyronin-Y are reported as −5.65 and −3.74 eV, respectively.

The extensive π-conjugation, strong fluorescence, and favorable electronic energy levels of both dyes make them excellent model analytes for investigating plasmon-enhanced optical phenomena. Their distinct molecular structures and electronic properties also enable a comparative assessment of the SERS performance of the synthesized Ag nanowires and ZnO nanoparticles.

3.3. Raman and photoluminescence analysis

The Raman and photoluminescence (PL) spectra of pyronin-Y and resorufin are shown in Fig. 8. Both molecules were excited using a 532 nm laser and exhibited strong fluorescence emission due to their highly conjugated molecular structures. As shown in Fig. 8a and b, the conventional Raman spectra are dominated by a broad fluorescence background, which masks most of the vibrational features and results in weak, poorly resolved Raman bands.

Fig. 8. Raman, and photoluminescence (a) and (c) pyronin-Y (b) and (d) resorufin molecules.

Fig. 8

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The intense fluorescence observed for both dyes present a significant challenge for Raman detection,41 as the fluorescence signal is several orders of magnitude stronger than the Raman scattering signal. Although increasing the acquisition time can improve the signal-to-noise ratio, prolonged laser exposure may lead to photodegradation of the dye molecules. Therefore, surface-enhanced Raman scattering (SERS) substrates based on Ag nanowires and ZnO nanoparticles were employed to enhance the Raman response and improve the detection of molecular vibrational modes.42

The PL spectra of pyronin-Y and resorufin (Fig. 8c and d) exhibit strong emission centered near 537 nm upon excitation at 532 nm. The small Stokes shift between the excitation and emission wavelengths indicates resonant fluorescence behavior and efficient radiative relaxation processes in both molecules.43 The high fluorescence intensity confirms their suitability as fluorescent probe molecules while also highlighting the need for plasmonic enhancement to obtain distinct Raman signatures.

Overall, the Raman and PL results demonstrate that pyronin-Y and resorufin are strongly fluorescent dyes with emission characteristics close to the excitation wavelength. Their pronounced fluorescence background makes them excellent model analytes for evaluating the SERS performance of the synthesized Ag nanowires and ZnO nanoparticles, where enhanced electromagnetic and charge-transfer effects can significantly improve the detection of vibrational fingerprints.

3.4. Surface-enhanced Raman scattering (SERS) performance of Ag and ZnO nanostructures

The SERS performance of the biosynthesized Ag nanowires (Ag NWs) and ZnO nanoparticles (ZnO NPs) was evaluated using the resonant fluorescent dyes resorufin and pyronin-Y as probe molecules. SERS spectra (Fig. 9) exhibited significantly enhanced and well-resolved vibrational bands, demonstrating the effectiveness of both nanostructured substrates in suppressing fluorescence interference and amplifying Raman signals.

Fig. 9. SERS spectra of resonant fluorescent molecules on Ag NPs and ZnO NPs exhibit enhanced vibrational signals; (a) Ag@resorufin (b) ZnO@resorufin (c) Ag@pyronin-Y (d) Ag@pyronin-Y.

Fig. 9

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For resorufin, characteristic vibrational bands were observed at 465, 579, 1173, 1327, 1413, 1487, 1644, and 2831 cm−1, corresponding to aromatic ring deformations, C–C, C–N, C Created by potrace 1.16, written by Peter Selinger 2001-2019 C, C Created by potrace 1.16, written by Peter Selinger 2001-2019 N, C Created by potrace 1.16, written by Peter Selinger 2001-2019 O, and C–H stretching vibrations.44 Similarly, the SERS spectrum of pyronin-Y displayed prominent bands at 1095, 1668, and 2831 cm−1, which are assigned to C–C/C Created by potrace 1.16, written by Peter Selinger 2001-2019 C skeletal vibrations and C–H stretching modes.45 The appearance and enhancement of these bands confirm strong interactions between the dye molecules and the nanoparticle surfaces, enabling sensitive detection of their molecular fingerprints.

A comparative analysis of the SERS performance (Fig. 10) demonstrated that ZnO NPs provided stronger Raman signal enhancement than Ag NWs for both probe molecules. Notably, the Raman intensity at 2831 cm−1 increased from 2270 a.u. on the Ag NW substrate to 3670 a.u. on the ZnO NP substrate (Fig. 10b). This enhanced performance may be attributed to the porous morphology, higher surface area, and defect-assisted charge-transfer interactions of the ZnO nanoparticles,46 which facilitate stronger analyte–substrate interactions and improved Raman signal amplification.

Fig. 10. Comparative Raman signal analysis of Ag and ZnO NPs for (a) resorufin and (b) pyronin-Y.

Fig. 10

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3.4.1. Substrate reproducibility and uniformity analysis

The reproducibility and uniformity of the synthesized SERS substrates were evaluated by recording Raman spectra from ten randomly selected positions on each substrate under identical experimental conditions. Relative standard deviation (RSD) values were calculated from the intensities of the characteristic Raman peaks of resorufin and pyronin-Y, and the results are summarized in Table 1.

Table 1. Reproducibility and uniformity analysis of SERS-substrates based on relative standard deviation.
Substrate Analyte Characteristic Raman peak (cm−1) Number of measurement spots (n) Mean intensity (a.u.) Standard deviation (±) RSD (%)
Ag NPs Resorufin 1413 10 1250 132 10.56
ZnO NPs Resorufin 1413 10 2485 168 6.76
Ag NPs Pyronin-Y 1668 10 980 101 10.31
ZnO NPs Pyronin-Y 1668 10 2015 145 7.19
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The ZnO substrates exhibited lower RSD values (6.76% for resorufin and 7.19% for pyronin-Y) compared to the Ag substrates (10.56% and 10.31%, respectively), indicating superior signal reproducibility and substrate uniformity. Moreover, low RSD values demonstrate that both materials provide stable and reproducible SERS enhancement. The comparatively lower RSD values of ZnO further highlight its potential as a reliable SERS substrate for the detection of highly fluorescent molecules.

3.4.2. Stability of green-synthesized SERS substrates

The long-term stability of the green-synthesized Ag nanowire (Ag NW) and ZnO nanoparticle (ZnO NP) SERS substrates was evaluated by monitoring the Raman signals of resorufin and pyronin-Y after storage under ambient laboratory conditions for different time periods.47 All measurements were performed under identical experimental conditions using characteristic Raman peaks as analytical markers.

Both substrates exhibited minimal variations in Raman intensity over time, indicating good stability and consistent SERS performance. However, the ZnO substrates showed superior signal retention compared to the Ag substrates, likely due to the higher chemical stability and oxidation resistance of ZnO nanostructures. In contrast, Ag nanowires are more susceptible to surface oxidation, which can gradually reduce their plasmonic enhancement efficiency.

The excellent stability of the ZnO substrates suggests that their porous morphology and defect-mediated charge-transfer interactions remain largely preserved during storage and repeated measurements.48 Furthermore, the reproducible Raman signals confirm the sustained adsorption capability and photon-scattering activity of the ZnO nanostructures.49 These results demonstrate that green-synthesized ZnO nanoparticles provide not only effective Raman enhancement but also superior long-term stability, making them promising SERS substrates for sustainable and reliable sensing applications.

The superior SERS performance of ZnO NPs can be attributed to a synergistic50 enhancement mechanism involving both photon scattering and charge-transfer interactions.51 The porous and rough surface morphology of ZnO increases light scattering and molecular adsorption, while intrinsic defects, particularly oxygen vacancies,52 generate surface-state energy levels that facilitate efficient charge transfer between the semiconductor surface and the adsorbed dye molecules. This chemical enhancement mechanism contributes substantially to Raman signal amplification (Fig. 11).53

Fig. 11. Comparison of peak intensities for (a) resorufin and (b) pyronin-Y molecules formed from Ag and ZnO nanoparticles.

Fig. 11

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In contrast, Ag NWs primarily enhance Raman scattering through the electromagnetic mechanism associated with localized surface plasmon resonance (LSPR). Although plasmonic enhancement is effective, it appears less efficient than the combined scattering and charge-transfer processes occurring in ZnO for these highly fluorescent molecules. Table 2 compares the vibrational signal data for the two molecules observed with Ag and ZnO nanoparticles in the SERS experiments.

Table 2. Experimental SERS wavenumbers of the assigned resorufin and pyronin molecular vibrational modes54–56.
Bond type SERS shift (cm−1)
Resorufin Pyronin-Y
In-plane bending of an aromatic ring 465 cm−1 —
C–H out-of-plane bending vibration 579 cm−1 —
C–C 1173 cm−1 1095 cm−1
C–N 1327 cm−1 —
C Created by potrace 1.16, written by Peter Selinger 2001-2019 C 1413 cm−1 1668 cm−1
C Created by potrace 1.16, written by Peter Selinger 2001-2019 N 1487 cm−1 —
C Created by potrace 1.16, written by Peter Selinger 2001-2019 O 1644 cm−1 —
C–H (sp2 hybridized) 2831 cm−1 2831 cm−1
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Overall, the results demonstrate that both Ag NWs and ZnO NPs function as effective SERS substrates; however, ZnO nanoparticles provide superior signal enhancement for resorufin and pyronin-Y. The enhanced performance is attributed to their high surface area, defect-assisted charge-transfer capability, and efficient light-scattering properties, highlighting their potential for ultrasensitive detection of fluorescent organic molecules.

3.5. SERS enhancement mechanism

The proposed charge-transfer (CT) mechanism is illustrated in Fig. 12. The energy levels of the conduction band edge (ECB) and the valence band edge (EVB) of ZnO are calculated via the equations ECB = −χ + 0.5 × Eg and EVB = ECB − Eg, respectively, where χ represents the absolute electronegativity of ZnO, which is 5.79 eV, as determined via theoretical calculations. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels of resorufin are −4.38 and −2.74 eV, respectively, while those of pyronin-Y are −5.65 and −3.74 eV.

Fig. 12. Charge-transfer mechansim of resorufin and pyronin-Y molecules with a ZnO substrate.

Fig. 12

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Under excitation with a 532 nm laser (2.33 eV), efficient charge transfer can occur between the ZnO substrate and the adsorbed dye molecules. As shown in Fig. 12, electrons may be transferred through the pathways I and IV for resorufin and through pathways II, III, and IV for pyronin-Y. These charge-transfer processes facilitate molecular polarization and increase the Raman scattering cross-section, leading to significant enhancement of the Raman signals.57,58 The enhanced SERS activity of the ZnO substrate can be attributed not only to the favorable alignment of the molecular energy levels with the semiconductor band structure but also to the presence of surface electronic states and the relatively narrow band gap of the ZnO thin films, which promote photoinduced charge transfer.59,60 As a result, both resorufin and pyronin-Y exhibit substantially stronger Raman signals on the ZnO substrate than on the Ag substrate. Overall, the results demonstrate that ZnO is an effective semiconductor SERS substrate for resonant fluorescent molecules. The observed enhancement is predominantly governed by the chemical enhancement (CE) mechanism through interfacial charge transfer between the ZnO thin film and the adsorbed dye molecules.

4. Conclusions

In this study, environmentally friendly Ag nanowires (Ag NWs) and ZnO nanoparticles (ZnO NPs) were successfully synthesized using Azadirachta indica leaf extract and evaluated as SERS substrates for the detection of the highly fluorescent dyes resorufin and pyronin-Y. Structural characterization confirmed the formation of phase-pure FCC Ag NWs and hexagonal wurtzite ZnO NPs, with average particle sizes of 86.11 nm and 26.15 nm, respectively. The synthesized nanostructures effectively suppressed fluorescence interference and enabled the observation of well-resolved Raman fingerprints of both analytes.

Comparative SERS investigations revealed that ZnO nanoparticles exhibited significantly higher enhancement efficiency than Ag nanowires, producing approximately two-fold stronger Raman signals for both resorufin and pyronin-Y. Characteristic vibrational bands corresponding to aromatic ring deformations, C–C, C–N, C Created by potrace 1.16, written by Peter Selinger 2001-2019 C, C Created by potrace 1.16, written by Peter Selinger 2001-2019 N, and C Created by potrace 1.16, written by Peter Selinger 2001-2019 O vibrations were clearly detected, demonstrating the capability of the synthesized substrates to recover Raman information otherwise masked by intense fluorescence. The superior performance of ZnO was attributed to its porous morphology, high surface area, enhanced photon scattering, and defect-assisted charge-transfer interactions. Energy-level analysis further confirmed that favorable band alignment between ZnO and the dye molecules facilitates efficient photoinduced charge transfer under 532 nm excitation, leading to substantial chemical enhancement of the Raman response.

These findings demonstrate that green-synthesized ZnO nanoparticles can serve as highly effective, low-cost, and environmentally benign alternatives to conventional noble-metal SERS substrates for the analysis of resonant fluorescent molecules. The combination of fluorescence quenching, strong Raman enhancement, and charge-transfer-assisted sensing highlights the potential of ZnO-based nanostructures for advanced applications in biosensing, molecular diagnostics, environmental monitoring, and optical spectroscopy. Furthermore, this work provides valuable insight into semiconductor-mediated SERS mechanisms and offers a sustainable strategy for developing next-generation sensing platforms.

Conflicts of interest

There is no conflicts to declare.

Acknowledgments

The authors would also like to thank Ongoing Research Funding Program (ORF-2026-668), King Saud University, Riyadh, Saudi Arabia.

Data availability

Data will be available upon request.

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