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<p>The generation of label-free and ultrasensitive biosensors for protein biomarker detection is still a significant goal in clinical diagnostics. In this paper, we report a Surface-Enhanced Raman Scattering (SERS) substrate using 3D Au–Ag nanopillars with tunable nanogap hotspots for the multiplexing and ultra-sensitive detection of protein biomarkers. The substrate, developed through sequential metal coating and nanolithography patterning, consisted of vertically aligned nanopillars with precisely controlled surface roughness and inter-pillar distances. Although optical characterizations and FDTD simulations proved broadband plasmonic resonance and strong electromagnetic field enhancement concentrated at the nanopillar nanogaps, structural and morphological studies (SEM, AFM, and EDS) confirmed uniform nanofeatures. Taking the C-reactive protein (CRP) as an example biomarker, the system exhibited excellent sensitivity and reproducibility with a detection limit as low as 1 femtomolar, an enhancement factor of ~ 3.2 × 10⁶, and a relative standard deviation of 7.8%. Such a small deviation is caused by intrinsic asperities of nanoscales due to the sequential deposition of Ag and Au, and a lack of minor deviations in hotspots triggering instead of by lithographic flaws. Although nanoimprint lithography (NIL) provides large-area stability, molecular assembly and localized plasmon coupling in nanogaps are stochastic and lead to the observed RSD. The label-free dual-analyte discriminating ability of the platform was proved by the multiplex detection of CRP and bovine serum albumin (BSA), which was achieved with easily resolved vibration peaks. Improved performance under both single and multiple detection modes was further evidenced through standard comparisons against existing SERS substrates. The platform also evidenced 28-day stability under ambient storage conditions, indicating promising durability for biosensing applications.</p> Graphical Abstract <p>This study illustrates the ultrasensitive detection of biomarkers by a 3D Ag-Au nanopillar SERS platform. The structure of the nanopillar substrate is illustrated on the left, highlighting the two metallic layers required for plasmonic amplification. The biomolecular interaction and laser stimulation of SERS detection are shown in the middle part. The multiplexing ability is seen on the right side by simultaneously binding CRP and BSA onto the surface. The ability of the system to differentiate between multiple proteins with high sensitivity and specificity is demonstrated by the Raman spectra, which give distinct, easily recognizable vibration peaks for each analysis.</p> <p></p>

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Ultrasensitive Detection and Spectral Analysis of Protein Biomarkers Using 3D Nanopillar SERS Platforms with Tunable Hotspots

  • 期刊论文

  • 发表日期：

    2025年10月13日

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  • Karthikeyan N.,
  • Jajjara Bhargav,
  • Karthik S.,
  • Kavitha M. S.

摘要

The generation of label-free and ultrasensitive biosensors for protein biomarker detection is still a significant goal in clinical diagnostics. In this paper, we report a Surface-Enhanced Raman Scattering (SERS) substrate using 3D Au–Ag nanopillars with tunable nanogap hotspots for the multiplexing and ultra-sensitive detection of protein biomarkers. The substrate, developed through sequential metal coating and nanolithography patterning, consisted of vertically aligned nanopillars with precisely controlled surface roughness and inter-pillar distances. Although optical characterizations and FDTD simulations proved broadband plasmonic resonance and strong electromagnetic field enhancement concentrated at the nanopillar nanogaps, structural and morphological studies (SEM, AFM, and EDS) confirmed uniform nanofeatures. Taking the C-reactive protein (CRP) as an example biomarker, the system exhibited excellent sensitivity and reproducibility with a detection limit as low as 1 femtomolar, an enhancement factor of ~ 3.2 × 10⁶, and a relative standard deviation of 7.8%. Such a small deviation is caused by intrinsic asperities of nanoscales due to the sequential deposition of Ag and Au, and a lack of minor deviations in hotspots triggering instead of by lithographic flaws. Although nanoimprint lithography (NIL) provides large-area stability, molecular assembly and localized plasmon coupling in nanogaps are stochastic and lead to the observed RSD. The label-free dual-analyte discriminating ability of the platform was proved by the multiplex detection of CRP and bovine serum albumin (BSA), which was achieved with easily resolved vibration peaks. Improved performance under both single and multiple detection modes was further evidenced through standard comparisons against existing SERS substrates. The platform also evidenced 28-day stability under ambient storage conditions, indicating promising durability for biosensing applications.

Graphical Abstract

This study illustrates the ultrasensitive detection of biomarkers by a 3D Ag-Au nanopillar SERS platform. The structure of the nanopillar substrate is illustrated on the left, highlighting the two metallic layers required for plasmonic amplification. The biomolecular interaction and laser stimulation of SERS detection are shown in the middle part. The multiplexing ability is seen on the right side by simultaneously binding CRP and BSA onto the surface. The ability of the system to differentiate between multiple proteins with high sensitivity and specificity is demonstrated by the Raman spectra, which give distinct, easily recognizable vibration peaks for each analysis.