Silver Nanowire-Gold Thin-Film Hybrid SERS Substrates for Enhanced Detection and Discrimination of Breast Cancer Cells

1. Introduction

Breast cancer remains the most commonly diagnosed cancer worldwide and continues to pose a major public health challenge. In the United States alone, an estimated 321,910 new cases of invasive breast cancer are expected in 2026 [1]. Globally, breast cancer accounted for approximately 2.4 million new cases in 2026, making it the most frequently diagnosed cancer among women and a leading cause of cancer-related mortality, with a mortality rate of approximately 6.9% [2]. Early detection is critical for improving patient outcomes, and routine breast cancer screening has been shown to reduce mortality by up to one-third, with some studies reporting reductions of 40–50%. Current clinical screening and diagnostic modalities include mammography, ultrasonography, magnetic resonance imaging (MRI), and positron emission tomography (PET), which are routinely employed to identify tumors at early and more treatable stages.

Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful analytical technique for cancer diagnosis because of its ability to provide highly sensitive biochemical information from cells and tissues [3,4,5,6,7]. SERS has been successfully applied to distinguish normal and malignant tissues in multiple organ systems, including the lung [8], breast [9,10,11,12,13,14], esophagus [15,16,17], and stomach [18]. The technique enables label-free detection of biomolecular constituents such as nucleic acids, proteins, lipids, collagen, phospholipids, phenylalanine, saccharides, and water, providing comprehensive biochemical fingerprints associated with disease states. Compared with conventional diagnostic methods, SERS offers several advantages, including high sensitivity, excellent molecular specificity, minimal interference from aqueous environments, high spatial resolution, and the capability for non-destructive and non-invasive analyses [19,20,21,22,23,24,25,26]. These features make SERS particularly attractive for both live-cell studies and potential clinical applications.

The performance of a SERS system is strongly dependent on the properties of the plasmonic substrate used to generate electromagnetic enhancement. Noble metal nanostructures, particularly gold (Au) and silver (Ag), are widely used because their localized surface plasmon resonance can significantly amplify Raman scattering signals [27,28,29,30]. However, conventional nanoparticle-based substrates often suffer from poor reproducibility, high background signals, aggregation-related variability, and limited biocompatibility. Consequently, the development of robust and reproducible SERS substrates capable of generating highly concentrated electromagnetic “hot spots” remains an active area of research.

Recent studies have demonstrated the potential of advanced SERS substrates for breast cancer detection. Chen et al. identified protein- and collagen-related spectral features associated with breast cancer progression [31]. Pan et al. developed a silver nanoparticle/porous silicon Bragg mirror (AgNPs/PSB) substrate that enabled the detection of serum-derived breast cancer biomarkers through characteristic protein and collagen signatures [32]. Yang et al. reported a single silver nanowire (AgNW)-based SERS platform capable of probing molecular information from breast cancer cells [33], while a three-dimensional Au@Ag bimetallic nanosphere substrate was subsequently employed for metabolite analysis in breast cancer patients [34]. In addition, Plancarte et al. demonstrated the discrimination of breast cancer cells using functionalized silver nanoparticles deposited on Au substrates [35].

Several studies have identified phenylalanine-associated Raman bands as potential markers of breast cancer. Gold nanoparticle-coated quartz substrates have been employed for cancer cell analysis, where prominent Raman features were attributed to protein vibrations, including the C–H in-plane bending mode of substituted benzene rings in phenylalanine [36]. González et al. further investigated the variation of phenylalanine band intensity across different stages of breast cancer [37], while Feng et al. reported enhanced phenylalanine-related Raman signals in malignant esophageal tissues [38]. These observations suggest that phenylalanine-associated spectral features may serve as valuable biomarkers for cancer diagnosis. The sensitivity of SERS arises from the strong local electromagnetic fields generated around metallic nanostructures, producing signal enhancements of several orders of magnitude when target molecules are located near plasmonic “hot spots” [39].

Despite significant progress, challenges remain in achieving high signal enhancement, low background noise, and reproducible spectral responses from biological samples. In the present study, we developed and evaluated two AgNW-based SERS platforms for breast cancer cell detection. AgNWs were deposited onto glass substrates to form conductive nanowire networks, while a hybrid AgNW/Au substrate was fabricated by depositing AgNWs onto thermally evaporated Au thin films on silicon. The morphology of the resulting substrates was characterized using field-emission scanning electron microscopy (FESEM), and their SERS performance was assessed using a model Raman probe molecule. To demonstrate their biological applicability, breast cancer SKBR3 cells and non-cancerous HEK-293 cells were immobilized on the substrates and analyzed using SERS. The study aimed to determine whether AgNW-modified Au thin films could provide enhanced spectral sensitivity and facilitate the identification of breast cancer-specific Raman signatures.

2. Materials and Methods

2.1. Cell Lines and Cell Culture Methods

Human breast cancer SKBR3 cells (ATCC, HTB-30) and human embryonic kidney HEK-293 cells (ATCC, CRL-1573) were used in this study. SKBR3 cells were cultured in McCoy’s 5A Modified Medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 16600082), whereas HEK-293 cells were maintained in Eagle’s Minimum Essential Medium (EMEM) containing L-glutamine (Quality Biological, Gaithersburg, MD, USA; Cat. No. 112-018-101). All culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific; Cat. No. A3840101) and 1% antibiotic–antimycotic solution (Gibco; Cat. No. 15240062).

Cells were maintained in T-25 tissue culture flasks (VWR, Radnor, PA, USA; Cat. No. 10062-874) at 37 °C in a humidified incubator supplied with 5% CO₂. Upon reaching approximately 80–90% confluence, cells were detached using 0.25% trypsin–EDTA solution (VWR; Cat. No. L0154-0100). The harvested cells were washed twice with Hank’s Balanced Salt Solution (HBSS) lacking calcium, magnesium, and phenol red (Fisher Scientific, Hampton, NH, USA; Cat. No. MP091810554) by centrifugation and resuspension.

Cell concentration was determined using methylene blue staining and a hemocytometer. The cell density was adjusted to 3 × 10⁶ cells mL⁻¹. Prior to surface-enhanced Raman spectroscopy (SERS) measurements, cell suspensions were stored on ice and gently dispersed by repeated pipetting immediately before deposition onto the Ag nanowire (AgNW)-coated substrates.

2.2. Preparation of AgNW-Based SERS Substrates and Cell Immobilization

Glass microscope slides were sequentially cleaned with ethanol and deionized (DI) water and allowed to dry prior to use. Silver nanowire (AgNW) suspensions (ACS Material, Pasadena, CA, USA; AgNW-120, Cat. No. NWAG04W1) were deposited onto the glass slides by drop-casting 20 μL aliquots at three separate locations. The coated slides were dried on a hot plate at 100 °C for 1 h.

For fabrication of Au-coated substrates, a chromium/gold (Cr/Au) thin film was deposited onto silicon wafers using a thermal evaporation system (PVD75 Thermal Evaporator, Kurt J. Lesker Company, Jefferson Hills, PA, USA). A 10 nm chromium adhesion layer was first deposited from 99.998% pure chromium pellets (Cat. No. EVMCR48-J) at a deposition rate of 0.8 Å s⁻¹ onto p-type silicon wafers (100 orientation; resistivity 1–10 Ω·cm; diameter 76.2 mm; thickness 525 ± 25 μm). Subsequently, a gold layer of 70 nm thickness was deposited using 99.999% pure gold pellets (Cat. No. EVMAU50QXQ) at deposition rates ranging from 1 to 3 Å s⁻¹.

AgNWs were then deposited onto the fabricated Au/Cr-coated silicon substrates via the same drop-casting procedure described above and dried at 100 °C for 1 h. For SERS measurements, 10 μL of each cell suspension was deposited onto both AgNW-coated glass and AgNW-coated Au substrates and allowed to immobilize prior to spectral acquisition (Figure 1).

2.3. SERS and Morphological Characterization

SERS measurements were performed using a SENTERRA II Raman microscope (Bruker Optics, Billerica, MA, USA) equipped with a thermoelectrically cooled charge-coupled device (CCD) detector. A 785 nm laser was focused onto the sample through a 20× objective lens. Raman spectra were collected over the spectral range of 50–3000 cm⁻¹ with a spectral resolution of 4 cm⁻¹. Unless otherwise stated, spectra were acquired using a laser power of 25 mW and an integration time of 1 s.

For each experimental condition, measurements were obtained from five independent samples, and the reported spectra represent the average signal intensity. The SERS enhancement performance of the fabricated substrates was evaluated using 1-octanethiol (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. 8218610100) dissolved in ethanol as a probe molecule.

The morphology of the fabricated substrates was examined using field-emission scanning electron microscopy (FESEM; Hitachi S-4700, Hitachi High-Technologies, Tokyo, Japan). Samples (5 × 4 mm²) were mounted on aluminum stubs using conductive carbon tape. Imaging was performed at an accelerating voltage of 15 kV.

3. Results

3.1. Morphological Characterization of AgNW-Based Substrates

The morphology of the fabricated substrates was investigated using FESEM, and representative images are shown in Figure 2. The AgNW-coated glass substrate exhibited a densely interconnected nanowire network covering most of the glass surface (Figure 2A). Higher-magnification imaging revealed extensive nanowire overlap and junction formation throughout the film. At the boundary between the coated and uncoated regions, portions of the underlying glass substrate remained visible, indicating the presence of pores within the AgNW network (Figure 2B).

The Au thin film exhibited a smooth and uniform surface morphology prior to AgNW deposition (Figure 2C). Following AgNW deposition, a dense nanowire network was formed on the Au surface (Figure 2D). The interface between the AgNW layer and the Au film is shown in Figure 2E, confirming successful assembly of the nanowire network on the metallic substrate.

After cell immobilization, both single cells and cell aggregates were observed on the AgNW-coated surfaces (Figure 2F). The cells remained attached to the nanowire film and occupied a substantial portion of the available surface area.This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

3.2. Optical Characterization of Cell-Functionalized Substrates

Confocal microscopy was used to visualize HBSS controls, HEK-293 cells, and SKBR3 cells on the fabricated substrates. Representative images obtained from AgNW-coated glass substrates are presented in Figure 3, whereas images collected from Au and AgNW/Au substrates are shown in Figure 4.

Cells were readily distinguishable from HBSS-coated regions on all substrate types. Both isolated cells and cell aggregates were observed. HEK-293 cells generally appeared smaller than SKBR3 cells. The images further confirmed successful immobilization of cells on the SERS-active substrates and enabled selection of cell-containing regions for subsequent Raman measurements.

3.3. SERS Performance Evaluation Using 1-Octanethiol

The SERS performance of the fabricated substrates was evaluated using 1-octanethiol as a model analyte. Figure 5 compares Raman spectra collected from AgNW-coated and uncoated substrates.

A characteristic Raman peak at approximately 1120 cm⁻¹ was clearly observed on both AgNW/glass and AgNW/Au substrates (Figure 5A,B). In contrast, the corresponding uncoated glass and Au substrates produced either negligible Raman signals or substantially weaker responses. The AgNW/Au substrate exhibited an enhancement factor of approximately 1500 relative to the bare Au substrate.

3.4. SERS Analysis of Cells on AgNW/Glass Substrates

Representative SERS spectra of HBSS, HEK-293 cells, and SKBR3 cells collected from AgNW/glass substrates are shown in Figure 6. Common Raman bands were observed near 1001, 1046, 1230, and 1450 cm⁻¹.

The spectra of HBSS and HEK-293 cells showed similar spectral profiles. In contrast, SKBR3 cells exhibited an additional Raman band at approximately 1028 cm⁻¹. Additional differences were observed in the spectral region between 1200 and 1500 cm⁻¹.

3.5. SERS Analysis of Cells on Au and AgNW/Au Substrates

SERS measurements performed on bare Au films generated weak and poorly defined spectra for HBSS, HEK-293, and SKBR3 samples (Figure 7).

Following the deposition of AgNWs onto the Au film, substantially enhanced Raman signals were observed (Figure 8). Peaks at approximately 1001, 1230, and 1451 cm⁻¹ were detected in all samples. Distinct bands at approximately 1003, 1028, and 1418 cm⁻¹ were observed in SKBR3 cells.

4. Discussion

The FESEM characterization confirmed the successful fabrication of interconnected AgNW networks on both glass and Au-film substrates. Such interconnected architectures are advantageous for SERS because nanowire junctions serve as localized electromagnetic hot spots that significantly enhance the Raman signals through localized surface plasmon resonance effects [27,28,29,30,39]. The dense nanowire coverage observed on the Au-supported substrates is expected to promote stronger plasmonic coupling between neighboring AgNWs and the underlying Au film, thereby increasing the local electromagnetic field intensity and improving SERS performance.

Confocal microscopy demonstrated successful immobilization of both HEK-293 and SKBR3 cells on the fabricated substrates. The ability to visually distinguish cells from HBSS-coated regions enabled accurate selection of measurement locations for Raman analysis. This observation is particularly important because reliable cell localization is essential for reducing spectral variability and improving the reproducibility of cell-based SERS measurements.

The use of 1-octanethiol as a model analyte revealed that the AgNW coatings were primarily responsible for the observed signal enhancement. Bare glass and Au substrates generated either negligible or weak Raman signals, whereas the AgNW/Au hybrid substrate produced an approximately 1500-fold enhancement of its intensity relative to the bare Au film. These results are consistent with previous studies demonstrating that Ag nanostructures provide stronger SERS enhancement than planar metallic films due to the formation of nanoscale hot spots at nanowire junctions and interparticle gaps [27,28,29,30,33,39]. Furthermore, the enhanced performance of the AgNW/Au substrate suggests that coupling between the AgNW network and the conductive Au support contributes to improved electromagnetic enhancement compared with AgNWs deposited on glass alone.

The SERS spectra obtained from SKBR3 breast cancer cells exhibited several characteristic Raman peaks that were absent or significantly weaker in HEK-293 cells and HBSS controls. Most notably, the Raman band observed at approximately 1028 cm⁻¹ can be attributed to C–H deformation vibrations associated with phenylalanine-containing proteins and collagen-related components [34,36,37,38]. Phenylalanine-associated Raman bands have previously been identified as important biomarkers in breast cancer and other malignancies [36,37,38]. Similarly, the band at approximately 1003 cm⁻¹ corresponds to the symmetric ring-breathing mode of phenylalanine, while the peak near 1230 cm⁻¹ has been assigned to tryptophan-associated vibrational modes [42,43]. The Raman band observed at approximately 1418 cm⁻¹ is consistent with COO⁻ symmetric stretching vibrations and nucleic-acid-related components that have been associated with cellular metabolic activity and alterations in cancerous tissues [44,45].

The detection of these characteristic spectral signatures is consistent with previous reports demonstrating that SERS can differentiate malignant and non-malignant cells based on their biochemical composition [9,10,11,12,13,14,31,32,33,34,35]. Chen et al. reported protein- and collagen-related Raman signatures associated with breast cancer progression [31], while Yang et al. demonstrated the capability of AgNW-based SERS substrates to probe molecular information from breast cancer cells [33]. Likewise, Plancarte et al. successfully discriminated breast cancer cells using silver nanostructures deposited on Au substrates [35]. The agreement between the present findings and these earlier studies supports the validity of the observed SKBR3-specific biomarkers.

Importantly, the SKBR3 spectra acquired on AgNW/glass and AgNW/Au substrates exhibited highly similar peak positions, indicating that the detected Raman signatures arise from cellular biochemical constituents rather than substrate-derived artifacts. However, the AgNW/Au hybrid substrate provided substantially stronger signal enhancement and improved spectral definition compared with the AgNW/glass platform. The Au support also permitted the use of thinner AgNW coatings while maintaining high SERS activity, thereby reducing substrate heterogeneity and facilitating more reproducible measurements. In contrast, thicker AgNW coatings were required on glass substrates to minimize background contributions from the underlying substrate, which may complicate optical interrogation of small cells and reduce measurement consistency.

Taken together, the results demonstrate that the AgNW/Au hybrid architecture combines the strong plasmonic activity of silver nanowires with the conductivity and structural stability of Au thin films. This synergistic interaction produces enhanced Raman sensitivity and facilitates the discrimination of breast cancer cells from non-cancerous controls based on phenylalanine-, protein-, and nucleic-acid-related biomarkers. These characteristics make AgNW/Au hybrid substrates promising candidates for label-free cancer diagnostics and cellular sensing applications.

5. Conclusions

Uniform and reproducible AgNW networks were successfully fabricated on both glass and Au thin-film substrates, yielding SERS-active platforms capable of enhancing Raman signals from molecular and cellular targets. Using 1-octanethiol as a model analyte, the AgNW-modified substrates exhibited substantially greater signal intensity than the corresponding bare glass and Au substrates, confirming the effectiveness of the AgNW network in generating strong plasmonic enhancement.

The developed substrates were subsequently evaluated for the detection of biological samples, including HBSS medium, non-cancerous HEK-293 cells, and breast cancer SKBR3 cells. While SERS measurements acquired on bare Au films produced weak and poorly defined spectra, AgNW-coated glass and AgNW/Au hybrid substrates generated multiple well-resolved Raman bands associated with cellular biochemical components. Notably, a distinct Raman peak at approximately 1028 cm⁻¹ was consistently detected in SKBR3 cells but was absent in HEK-293 cells and HBSS controls. This feature is associated with phenylalanine-containing biomolecules and represents a potential spectral marker for breast cancer cell identification.

Although both AgNW/glass and AgNW/Au platforms enabled the detection of cancer-related spectral signatures, the AgNW/Au hybrid substrate demonstrated several practical advantages, including reduced spectral background, improved signal quality, and enhanced measurement reproducibility. These benefits are attributed to the synergistic interaction between the highly conductive Au thin film and the AgNW network, which promotes efficient plasmonic coupling and increased electromagnetic hot-spot density.

Overall, the results demonstrate that AgNW/Au hybrid thin-film substrates provide a robust and sensitive SERS platform for the label-free detection and discrimination of breast cancer cells. The ability to identify characteristic cancer-associated Raman signatures, combined with the enhanced signal amplification afforded by the hybrid architecture, highlights the potential of this platform for future applications in cancer diagnostics, biomarker discovery, and biomedical sensing. Future studies will focus on validating the approach using additional breast cancer cell lines and clinical specimens to further establish its diagnostic utility.