Surface Enhanced Raman Spectroscopy (SERS)

In this article I will give an overview of Surface Enhanced Raman Spectroscopy (SERS).

Figure 1 – Credit RSC review article DOI: 10.1039/D1NR00708D. Diagrams of the two SERS-based approaches for bioanalyte detection in spectral pathology. On the left, label-based SERS detection. On the right, label-free SERS detection.

What is SERS?

SERS allows detection of small amounts of materials with great precision. It is a type of Raman Spectroscopy that boosts signal strength millions of times using nanostructured metal surfaces like gold or silver. This makes it possible to detect and analyse even a single molecule. In metal nanostructures, molecules are placed on a rough metal surface. During localised surface plasmon resonance (LSPR), a light excites surface electrons in the metal, creating intense electromagnetic fields. For enhanced Raman Scattering the molecules near the metal surface experience an amplified Raman signal, making detecting ultra-sensitive.

Current uses

SERS is a mature field and has a lot of use cases, companies like Owlstone Medical use it for volatile organic compounds for non-invasive screening. It was also employed during the pandemic to detect SARS-CoV-2 and Nanoplex/SERS ID offer commercial kits for rapid SERS-based pathogen detection in biofluids.

SERS is used in biosensing and medical diagnostics to detect the diseases viruses and biomarkers. In Chemical and Environmental Analysis to identify pollutants and toxins at trace levels. In forensics and security to detect drugs explosives and counterfeit materials. In electrochemistry and catalysis to study reaction mechanisms at electrode interfaces.

Mechanisms and Theory

SERS combines nanotechnology, plasmonics and spectroscopy making it one of the most powerful analytical tools for ultrasensitive detection. Plasmonics is the study of how light interacts with the free electrons in metallic nanostructures, causing the electrons to oscillate collectively. This interaction creates wave-like excitations called plasmons, which can confine and manipulate light on the nanoscale.

Light Scattering

Scattering describes processes where particles or radiation are forced to deviate from their straight trajectory. There are three main domains of light scattering that can be distinguished by the relative size of the scattering particle P to the wavelength of the light λ (lambda).

Geometric Scattering P>> λ – Incoming angle = outgoing angle, weak wavelength dependence

Mie-Scattering P ~ λ – Asymmetric complex scattering behaviour, weak wavelength dependence

Rayleigh Scattering P<< λ – Symmetric scattering, strong wavelength dependence.

Fig. 2 – credit NDT4Industry – Talk Robert Zimmerleiter: SERS – Surface Enhanced Ramen Spectroscopy YouTube

Most photons get scattered elastically (no energy exchange with the sample. However, around 1 in 1 million, get scatter inelastically, so exchange energy with the sample. For inelastic scatters, light gets scatter with either lower energy (longer wavelength, Stokes scattering) or higher energy (shorter wavelength, Anti-Stokes scattering)

The amount of exchanged energy is wavelength dependent and sample dependent, thus giving a chemical fingerprint of the measured sample.

To measure energy loss of the inelastically scattered light (chemical fingerprint), initial energy E = hc/ λ must be known precisely. To get a good signal strength of inelastically scattered light (1 in 1 million) very high light intensity is required. So, a strong monochromatic light source is needed, thus a laser.

Surface Enhanced Raman Spectroscopy (SERS) uses surfaces of tiny metal structures (roughened surfaces, nanoparticles, nanotubes) for signal enhancement up to a factor of 10¹¹ (discovered in the 1970s) relative to normal Raman signal. SERS can even detect analytes at the femtomolar (fM, 10^-15 mols / litre) scale with optimised substrates.

There are two theories for the explanation of the effect that are still debated:

Electromagnetic Theory- Electric field enhancement via localized surface plasmon excitation (collective oscillations of free electrons in a metal nanoparticle, triggered when the nanoparticle’s size is similar to the wavelength of light)- Enhancements happens with incoming and scattered light à double enhancement- Surface plasmons appear on metal surfaces with near-zero band gaps

Chemical Theory- Resonance Raman spectroscopy explains the enhancements- High intensity charge transfers from metal surface to the adsorbing species- Effect is relevant for nanoclusters with considerable band gaps

Most likely both effects appear in combination.

Exploitation of SERS effect

To exploit the SERS-effect, the sample has to be in close contact with the nanostructure / Nanoparticles. There are two ways of doing this:

Liquid-SERS, is where the sample is dissolved in nanoparticle-solution and the Raman-measurement is conducted in the liquid, containing nanoparticles (NP) and the sample. Advantages, high laser power can be used for the measurement, signal strength is independent from measurement position inside the liquid. However, samples may not dissolve in the NP solution, if it does, it gets further diluted.

For SERS-substrates, samples are dropped on to the substrate with nanostructure/nanoparticles on its surface. Then Raman Spectroscopy is conducted on SERS substrate. Advantages, amount of backscattered light is larger than for liquid SERS, so sample does not get further diluted when applied to the substrate. Disadvantages, differences depending on measurement position are possible, some SERS-substrates can be damaged when high laser powers are applied.

A number of challenges remain for further applicability of SERS in biological and clinical environments. Different substrates strongly differ in terms of price, signal enhancement, ease of use, damage threshold, ideal laser wavelength, measurement reproducibility, background signal.

SERS for biomarker and disease analysis

Fig. 3 - Schematic illustration of four different SERS substrates for a variety of bioanalyte detection. (A) Klarite substrate for the detection of different pathogens. (B) Gold film over nanosphere substrate for bacterium and aptamer detection. (C) Vancomycin-coated Ag/anodic aluminum oxide (AAO)–SERS substrate for bacterium and blood cell detection. (D) Carboxylic-acid-functionalized and graphitic nanolayer-coated three-dimensional SERS substrate (CGSS) substrate for the detection of fibrils.

SERS activated platforms offer high sensitivity and promising multiplexing ability in bioanalysis and diseases diagnosis. There is diversity of SERS-based assays, including label-based and label-free approaches on microfluidic chips on paper based substrate.

The core strengths are:

- Its high sensitivity and rapid detection down to single cell or molecular levels.

- It enables quantitative fingerprinting of cells and tissues with high spatial resolution.

- It’s non-invasive with minimal sample prep making it ideal for clinical and point of care settings.

As well as spectral deconvolution to analyse a broad range of biomolecules such as lipids, DNA and proteins. Especially allowing targeted treatments for diseases like sepsis due to fast pathogen identification, this is a real-life saving aspect in treating sepsis.

SERS supports multiplexing or detecting multiple targets simultaneously via microfluidic chips, paper-based platforms and lab on a chip system.

Fig. 4 Prospects and challenges of SERS-based assays in bioanalysis and diagnosis

However, like all spectroscopic techniques there are challenges:

- It still has to overcome signal variability and reproducibility issues due to nano-optical instability and surface chemistry effects.

- It also requires control of electromagnetic hotspots and a deeper understanding of signal origins in complex samples.

- As the electromagnetic enhancement is the main contribution to the SERS signal, changes induced at the dielectric-metal interface, like controlled modification of refractive index are highly important.

- Background signal interference from biological matrices like blood or proteins- Quantitative SERS is still less robust than NS in complex fluids like serum or plasma unless highly controlled

AI

With data analysis and machine learning, AI can analyse complex spectra fast aiding with automated diagnosis. Future applications include next-generation surgery tools, real-time diagnostics, and large-scale biomarker screening. In addition, PCA (Principal Component Analysis) and LDA (Linear Discriminant Analysis) allow unsupervised and supervised methods that maximise data variance and separation between classes. As spectra can be interpreted as  images convolutional neural networks (CNN) can also be used.

Conclusion

Ultimately, a SERS database compromising most common and clinically relevant pathogens would spur data analysis by generating statistically relevant input data for the development of new multi-level clustering algorithms.