Plasmonic nanostructures have shown great potential for use in biosensing and single-molecule detection owing to their optical properties. Their localized surface plasmon resonances (LSPR) provide many advantages in analytical spectroscopic techniques.1 In surface enhanced Raman scattering (SERS), the Raman scattering signal of a molecule can be enhanced by several orders of magnitude due to high electromagnetic field enhancements as a consequence of the LSPR.2, 3 Very early work in SERS focused on electrochemical systems and simple analytes,4 while later studies identified the potential of plasmonic nanoparticles, particularly gold nanoparticles,5 as efficient SERS substrates for large molecules in biosensing applications.

SERS spectra of proteins,6-8 amino acids,9 nucleic acids and nucleic bases,3 10 and live cells11 have been reported using spherical gold nanoparticles. Anisotropic nanostructures such as nanorods and nanostars have also been used to create different types hot-spots that provide very high field enhancements.12, 13 More complex nanostructures like shell-isolated nanoparticles, DNA-hybridized nanostructures,14 immobilized nanoparticle substrates15 and nanoshell arrays16 have been reported to show high stability of substrate-analyte interactions and thereby selectivity for probing different modes in the vibrational SERS spectra. The detection of single DNA and protein molecules by SERS dates back for several decades, and includes the detection of single hemoglobin molecules,8 DNA bases,3 and amino acids.17 The field of molecular plasmonics, specifically the improved control of molecule-nanostructure interactions by positioning biomacromolecules in high local fields of plasmonic nanostructures, e.g., by the use of DNA origami technology, helped to improve the high sensitivity for detection of specific functional groups by SERS.18, 19

Plasmonic nanopores emerged as plasmonic substrates that confine the biomolecule-metal nanostructure interaction when the biomolecules translocate through them. Nanopore-based approaches were shown to be effective in distinguishing single molecule DNA bases and single amino acids in DNA and protein molecules, respectively.20, 21

However, the high fluctuation of SERS signals at high enhancement levels22 poses great challenges with respect to fully exploiting the vibrational information that is contained in the SERS data about specific molecular structure and interaction. As example, the concentration-dependent interactions of a protein with a gold nanostructure were shown to greatly depend on the concentration and sequence of a protein, even at high sequence similarity.6

As such, trapping of biomolecules in SERS hot spots while translocating them through plasmonic nanopores is an effective strategy to isolate a biomolecule of interest for SERS characterization.20, 23 This project focuses on the following main aspects to improve in this approach by implementing: An efficient temporal control of biomolecule translocation24 The design of optimized plasmonic and identification of excitation conditions that yield an actual vibrational structural characterization based on more than a few bands attained previously21 The interpretation of intrinsic variations that are present in any SERS spectra, to identify spectral fingerprints of different structures of biomolecule analytes, such as protein secondary structure.25

References

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• (3) Kneipp, K.; Kneipp, H.; Kartha, V. B.; Manoharan, R.; Deinum, G.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Detection and identification of a single DNA base molecule using surface-enhanced Raman scattering (SERS). Physical Review E 1998, 57 (6), R6281-R6284. DOI: 10.1103/PhysRevE.57.R6281.
• (4) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Raman spectra of pyridine adsorbed at a silver electrode. Chemical Physics Letters 1974, 26 (2), 163-166. DOI: https://doi.org/10.1016/0009-2614(74)85388-1.
• (5) Raschke, G.; Kowarik, S.; Franzl, T.; Sönnichsen, C.; Klar, T. A.; Feldmann, J.; Nichtl, A.; Kürzinger, K. Biomolecular Recognition Based on Single Gold Nanoparticle Light Scattering. Nano Letters 2003, 3 (7), 935-938. DOI: 10.1021/nl034223+.
• (6) Szekeres, G. P.; Kneipp, J. Different binding sites of serum albumins in the protein corona of gold nanoparticles. Analyst 2018, 143 (24), 6061-6068. DOI: 10.1039/c8an01321g From NLM Medline.
• (7) Szekeres, G. P.; Kneipp, J. SERS Probing of Proteins in Gold Nanoparticle Agglomerates. Front Chem 2019, 7, 30. DOI: 10.3389/fchem.2019.00030 From NLM PubMed-not-MEDLINE.
• (8) Xu, H.; Bjerneld, E. J.; Käll, M.; Börjesson, L. Spectroscopy of Single Hemoglobin Molecules by Surface Enhanced Raman Scattering. Physical Review Letters 1999, 83 (21), 4357-4360. DOI: 10.1103/PhysRevLett.83.4357.
• (9) Podstawka, E.; Ozaki, Y.; Proniewicz, L. M. Part III: Surface-Enhanced Raman Scattering of Amino Acids and Their Homodipeptide Monolayers Deposited onto Colloidal Gold Surface. Applied Spectroscopy 2005, 59 (12), 1516-1526. DOI: 10.1366/000370205775142520 (acccessed 2024/06/11).
• (10) Peticolas, W. L. Raman spectroscopy of DNA and proteins. In Methods in Enzymology, Vol. 246; Academic Press, 1995; pp 389-416.
• (11) Živanović, V.; Milewska, A.; Leosson, K.; Kneipp, J. Molecular Structure and Interactions of Lipids in the Outer Membrane of Living Cells Based on Surface-Enhanced Raman Scattering and Liposome Models. Analytical Chemistry 2021, 93 (29), 10106-10113. DOI: 10.1021/acs.analchem.1c00964.
• (12) Spedalieri, C.; Szekeres, G. P.; Werner, S.; Guttmann, P.; Kneipp, J. Intracellular optical probing with gold nanostars. Nanoscale 2021, 13 (2), 968-979, 10.1039/D0NR07031A. DOI: 10.1039/D0NR07031A.
• (13) Fazio, B.; D’Andrea, C.; Foti, A.; Messina, E.; Irrera, A.; Donato, M. G.; Villari, V.; Micali, N.; Maragò, O. M.; Gucciardi, P. G. SERS detection of Biomolecules at Physiological pH via aggregation of Gold Nanorods mediated by Optical Forces and Plasmonic Heating. Scientific Reports 2016, 6 (1), 26952. DOI: 10.1038/srep26952.
• (14) Heck, C.; Prinz, J.; Dathe, A.; Merk, V.; Stranik, O.; Fritzsche, W.; Kneipp, J.; Bald, I. Gold Nanolenses Self-Assembled by DNA Origami. ACS Photonics 2017, 4 (5), 1123-1130. DOI: 10.1021/acsphotonics.6b00946.
• (15) Cheng, H.-W.; Huan, S.-Y.; Wu, H.-L.; Shen, G.-L.; Yu, R.-Q. Surface-Enhanced Raman Spectroscopic Detection of a Bacteria Biomarker Using Gold Nanoparticle Immobilized Substrates. Analytical Chemistry 2009, 81 (24), 9902-9912. DOI: 10.1021/ac9014275.
• (16) Wang, H.; Kundu, J.; Halas, N. J. Plasmonic Nanoshell Arrays Combine Surface-Enhanced Vibrational Spectroscopies on a Single Substrate. Angewandte Chemie International Edition 2007, 46 (47), 9040-9044. DOI: https://doi.org/10.1002/anie.200702072 (acccessed 2026/05/12).
• (17) Brulé, T.; Yockell-Lelièvre, H.; Bouhélier, A.; Margueritat, J.; Markey, L.; Leray, A.; Dereux, A.; Finot, E. Sorting of Enhanced Reference Raman Spectra of a Single Amino Acid Molecule. The Journal of Physical Chemistry C 2014, 118 (31), 17975-17982. DOI: 10.1021/jp504395c.
• (18) Tapio, K.; Mostafa, A.; Kanehira, Y.; Suma, A.; Dutta, A.; Bald, I. A Versatile DNA Origami-Based Plasmonic Nanoantenna for Label-Free Single-Molecule Surface-Enhanced Raman Spectroscopy. ACS Nano 2021, 15 (4), 7065-7077. DOI: 10.1021/acsnano.1c00188.
• (19) Heck, C.; Kanehira, Y.; Kneipp, J.; Bald, I. Placement of Single Proteins within the SERS Hot Spots of Self-Assembled Silver Nanolenses. Angewandte Chemie International Edition 2018, 57 (25), 7444-7447. DOI: https://doi.org/10.1002/anie.201801748 (acccessed 2026/05/15).
• (20) Huang, J.-A.; Mousavi, M. Z.; Zhao, Y.; Hubarevich, A.; Omeis, F.; Giovannini, G.; Schütte, M.; Garoli, D.; De Angelis, F. SERS discrimination of single DNA bases in single oligonucleotides by electro-plasmonic trapping. Nature Communications 2019, 10 (1), 5321. DOI: 10.1038/s41467-019-13242-x.
• (21) Huang, J.-A.; Mousavi, M. Z.; Giovannini, G.; Zhao, Y.; Hubarevich, A.; Soler, M. A.; Rocchia, W.; Garoli, D.; De Angelis, F. Multiplexed Discrimination of Single Amino Acid Residues in Polypeptides in a Single SERS Hot Spot. Angewandte Chemie International Edition 2020, 59 (28), 11423-11431. DOI: https://doi.org/10.1002/anie.202000489 (acccessed 2026/05/12).
• (22) Lindquist, N. C.; de Albuquerque, C. D. L.; Sobral-Filho, R. G.; Paci, I.; Brolo, A. G. High-speed imaging of surface-enhanced Raman scattering fluctuations from individual nanoparticles. Nature Nanotechnology 2019, 14 (10), 981-987. DOI: 10.1038/s41565-019-0535-6.
• (23) Crick, C. R.; Albella, P.; Kim, H.-J.; Ivanov, A. P.; Kim, K.-B.; Maier, S. A.; Edel, J. B. Low-Noise Plasmonic Nanopore Biosensors for Single Molecule Detection at Elevated Temperatures. ACS Photonics 2017, 4 (11), 2835-2842. DOI: 10.1021/acsphotonics.7b00825.
• (24) Di Fiori, N.; Squires, A.; Bar, D.; Gilboa, T.; Moustakas, T. D.; Meller, A. Optoelectronic control of surface charge and translocation dynamics in solid-state nanopores. Nature Nanotechnology 2013, 8 (12), 946-951. DOI: 10.1038/nnano.2013.221.
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In the last three years during, there have been several developments in the field of plasmon-enhanced nanospectroscopy of biomolecules using a variety of plasmonic substrates. For proteins in particular, plasmonic nanopores were applied to detect single proteins by both electrical and optical sensing.1, 2 The dwell time or amount of time the protein molecule is trapped in the pore for detection was improved by several new approaches. SERS combined with a nanopore was used for identification of single amino acid substitutions in peptides,3 the extraction of prominent spectral signatures of protein structure from SERS spectra4 and single protein molecule fingerprinting.5 Analysis of changes in the structural conformation of proteins translocating through a nanopore were reported.6, 7 DNA origami engineered nanostructures which were able to control the SERS hot-spots and improve SERS detection limit have also been reported.8-10

Novel plasmonic substrates have been developed in the past three years which were successfully applied to study biomolecules using spectroscopic techniques. These make use of traditional plasmonic metals like gold and silver, as well as new-generation plasmonic materials based on rhodium and aluminium nanostructures. Complex substrates like rhodium nanoparticles in nanoporous aluminium in the DYNAMO project,11 gold nanbipyramid-silver nanorod structures,12 layered bimetal nanoporous platforms13 and bimetallic nanostars14 have been developed for use in biomolecule SERS. Our group reported non-resonant surface enhanced hyper Raman scattering (SEHRS) spectra of amino acids recently, using gold nanoparticles, which provides complementary information to the spectral data obtained from SERS of amino acids.15 Effect of variations in physico-chemical conditions in SERS spectra of different biomolecules were also investigated such as change in temperature,16 differences in excitation wavelength and several others.

Simultaneously, there has been several new developments in the analysis and processing of spectroscopic data with machine learning techniques being used to extract important information from electrical and optical data by us and other groups. These techniques have been applied for molecular quantification,17 single molecule discrimination of a proline modification using deep learning,18 and broader integration of artificial intelligence in biosensing platforms.19 Both supervised and unsupervised learning techniques to study spectroscopic data have improved.20, 21 Specifically, classification models like partial least squares – discriminant analysis (PLS-DA) and random forests (RF) have been reported for their usefulness in both classification as well as analysis of SERS data from other biomolecules by our lab that are currently being adapted to the analysis of few-molecule SERS spectra of proteins as well.22, 23

References

• (1) Yu, L.; Kang, X.; Li, F.; Mehrafrooz, B.; Makhamreh, A.; Fallahi, A.; Foster, J. C.; Aksimentiev, A.; Chen, M.; Wanunu, M. Unidirectional single-file transport of full-length proteins through a nanopore. Nature Biotechnology 2023, 41 (8), 1130-1139. DOI: 10.1038/s41587-022-01598-3.
• (2) Sauciuc, A.; Morozzo della Rocca, B.; Tadema, M. J.; Chinappi, M.; Maglia, G. Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force. Nature Biotechnology 2024, 42 (8), 1275-1281. DOI: 10.1038/s41587-023-01954-x.
• (3) Li, W.; Guo, L.; Ding, X.-L.; Ding, Y.; Ji, L.-N.; Xia, X.-H.; Wang, K. High-Throughput Single-Molecule Surface-Enhanced Raman Spectroscopic Profiling of Single-Amino Acid Substitutions in Peptides by a Gold Plasmonic Nanopore. ACS Nano 2024, 18 (29), 19200-19207. DOI: 10.1021/acsnano.4c04775.
• (4) Zhou, J.; Lan, Q.; Li, W.; Ji, L.-N.; Wang, K.; Xia, X.-H. Single Molecule Protein Segments Sequencing by a Plasmonic Nanopore. Nano Letters 2023, 23 (7), 2800-2807. DOI: 10.1021/acs.nanolett.3c00086.
• (5) Soni, N.; Rosenstock, Z.; Verma, N. C.; Siddharth, K.; Talor, N.; Liu, J.; Marom, B.; Kolomeisky, A. B.; Aksimentiev, A.; Meller, A. Full-length protein classification via cysteine fingerprinting in solid-state nanopores. Nature Nanotechnology 2025, 20 (10), 1482-1490. DOI: 10.1038/s41565-025-02016-w.
• (6) Zhou, J.; Gao, C.; Ding, Y.; Nie, Z.; Xu, M.; Fu, P.; He, B.; Wang, S.; Xia, X.-H.; Wang, K. Multidimensional Investigations of Single Molecule Unfolding of Bovine Serum Albumin Using Plasmonic Nanopores. Nano Letters 2025, 25 (15), 6325-6331. DOI: 10.1021/acs.nanolett.5c01214.
• (7) Ding, Y.; Li, W.; Wang, Z.; Chen, J.; Xia, X.-H.; Ji, L.-N.; Zhou, J.; Wang, K. Real-Time SERS Tracking of Single-Protein Orientational Change via Plasmonic Nanopore Confinement. Analytical Chemistry 2025, 97 (48), 26532-26538. DOI: 10.1021/acs.analchem.5c04818.
• (8) Wang, Y.; Cai, Y.; Jin, H.; Jin, S. DNA origami-engineered gold nanoparticle multimers for ultrasensitive, label-free SERS detection of small molecules and biomolecules. RSC Advances 2026, 16 (23), 21266-21276, 10.1039/D6RA02441F. DOI: 10.1039/D6RA02441F.
• (9) Sharma, M.; Kaur, C.; Singhmar, P.; Rai, S.; Sen, T. DNA origami-templated gold nanorod dimer nanoantennas: enabling addressable optical hotspots for single cancer biomarker SERS detection. Nanoscale 2024, 16 (32), 15128-15140, 10.1039/D4NR01110D. DOI: 10.1039/D4NR01110D.
• (10) Schuknecht, F.; Kołątaj, K.; Steinberger, M.; Liedl, T.; Lohmueller, T. Accessible hotspots for single-protein SERS in DNA-origami assembled gold nanorod dimers with tip-to-tip alignment. Nature Communications 2023, 14 (1), 7192. DOI: 10.1038/s41467-023-42943-7.
• (11) Banerjee, S.; Mattarozzi, L.; Maccaferri, N.; Cattarin, S.; Weng, S.; Douaki, A.; Lanzavecchia, G.; Sapunova, A.; D'Amico, F.; Ma, Q.; et al. Porous aluminum decorated with rhodium nanoparticles: preparation and use as a platform for UV SERS. Materials Advances 2024, 5 (15), 6248-6254, 10.1039/D4MA00203B. DOI: 10.1039/D4MA00203B.
• (12) Luan, L.; Zhang, X.; Li, P.; Xu, W. SERS substrate based on large-scale self-assembled Au nanobipyramid@Ag nanorod multifunctional paper-based materials for practical and reliable quantitative SERS detection. Analytical and Bioanalytical Chemistry 2025, 417 (13), 2903-2913. DOI: 10.1007/s00216-025-05830-2.
• (13) Zhang, N.; Sreekanth, K. V.; Chen, Y. F.; Teo, S. L.; Ke, L.; Zhao, M.; Teng, J. Scalable Multilayered Plasmonic Nanoporous Films for Surface-Enhanced Raman Spectroscopy. ACS Applied Optical Materials 2024, 2 (5), 744-749. DOI: 10.1021/acsaom.4c00048.
• (14) Negrín-Montecelo, Y.; Elsaidy, A.; Giráldez-Martínez, J.; Carbó-Argibay, E.; Wang, Z.; Govorov, A. O.; Alvarez-Puebla, R. A.; Correa-Duarte, M. A.; Besteiro, L. V. Unveiling multimodal hot carrier excitation in plasmonic bimetallic Au@Ag nanostars for photochemistry and SERS sensing. Nano Research 2024, 17 (12), 10355-10362. DOI: 10.1007/s12274-024-6950-5.
• (15) Rahman, Fariha B.; Kneipp, J. Nonresonant Surface-Enhanced Hyper Raman Scattering of Aromatic Amino Acids on Gold Nanoparticles. Journal of Raman Spectroscopy 2026, n/a (n/a). DOI: https://doi.org/10.1002/jrs.70106 (acccessed 2026/05/15).
• (16) Klenotová, M.; Matějka, P. Investigating temperature-dependent spectral changes in human saliva using SERS on Ag and Au surfaces. Vibrational Spectroscopy 2025, 138, 103788. DOI: https://doi.org/10.1016/j.vibspec.2025.103788.
• (17) Bi, X.; Czajkowsky, D. M.; Shao, Z.; Ye, J. Digital colloid-enhanced Raman spectroscopy by single-molecule counting. Nature 2024, 628 (8009), 771-775. DOI: 10.1038/s41586-024-07218-1.
• (18) Zhao, Y.; Zhan, K.; Xin, P.-L.; Chen, Z.; Li, S.; De Angelis, F.; Huang, J.-A. Single-Molecule SERS Discrimination of Proline from Hydroxyproline Assisted by a Deep Learning Model. Nano Letters 2025, 25 (18), 7499-7506. DOI: 10.1021/acs.nanolett.5c01177.
• (19) Ebrahimi, F.; Kumari, A.; Dellinger, K. Integration of Nanoengineering with Artificial Intelligence and Machine Learning in Surface-Enhanced Raman Spectroscopy (SERS) for the Development of Advanced Biosensing Platforms. Advanced Sensor Research 2025, 4 (2), 2400155. DOI: https://doi.org/10.1002/adsr.202400155 (acccessed 2026/05/15).
• (20) Jensen, M. N.; Guerreiro, E. M.; Enciso-Martinez, A.; Kruglik, S. G.; Otto, C.; Snir, O.; Ricaud, B.; Hellesø, O. G. Identification of extracellular vesicles from their Raman spectra via self-supervised learning. Scientific Reports 2024, 14 (1), 6791. DOI: 10.1038/s41598-024-56788-7.
• (21) Xu, G.; Bao, Y.; Zhang, Y.; Xiang, X.; Luo, H.; Guo, X. Applying Machine Learning and SERS for Precise Typing of DNA Secondary Structures. Analytical Chemistry 2024, 96 (43), 17109-17117. DOI: 10.1021/acs.analchem.4c02143.
• (22) Feng, Y.; Gärber, F.; Saied, E. M.; Spedalieri, C.; Kochovski, Z.; Werner, S.; Pratsch, C.; Arenz, C.; Seifert, S.; Kneipp, J. SERS Spectra Indicate the Molecular Effects of 7-Nitrobenz-2-oxa-1,3-diazole (NBD) on Living Cells. The Journal of Physical Chemistry C 2024, 128 (46), 19722-19735. DOI: 10.1021/acs.jpcc.4c05260.
• (23) Feng, Y.; Gärber, F.; Saied, E. M.; Singh, H.; Spedalieri, C.; Werner, S.; Pratsch, C.; Arenz, C.; Seifert, S.; Kneipp, J. Endolysosomal Impact of Elevated Ceramide Levels Revealed by Optical and Ultrastructural Nanoprobing. ACS Nano 2026. DOI: 10.1021/acsnano.5c22213.