Thiol-polyethylene glycol-folic acid (HS-PEG-FA) induced aggregation of Au@Ag nanoparticles: A SERS and extinction UV-Vis spectroscopy combined study.

Plasmonic colloidal nanoparticles (NPs) functionalised with polymers are widely employed in diverse applications, offering advantages demonstrated over non-functionalised NPs such as enhanced colloidal stability or increased biocompatibility. However, functionalisation with polymers does not always increase the stability of the colloidal system. This work explores the intricate relationship between the functionalisation of plasmonic core@shell Au@Ag nanoparticles (NPs) with thiol-polyethylene glycol-folic acid (HS-PEG-FA) polymer chains and the resulting stability and spectral characteristics of Surface-Enhanced Raman Scattering (SERS) nanotags based on these NPs. We demonstrate that varying levels of HS-PEG-FA grafting influence nanotag stability, with a low level of grafting causing aggregation and subsequently affecting the spectral signature of Raman-reporter molecules attached to the surface of the NP. Electrostatic destabilisation is identified as the primary mechanism driving aggregation, impacting the SERS spectrum of Malachite Green isothiocyanate (MGITC) whose spectral shape is different between the aggregated and non-aggregated NPs. The findings provide valuable insights into NPs stability under different conditions, offering essential considerations for the design and optimisation of SERS nanotags in bio-analytical applications, particularly those involving data processing based on spectral shape, such as in multiplex approaches where experimental spectra are decomposed with several reference components.

Designing SERS nanotags for profiling overexpressed surface markers on single cancer cells: A review.

This review focuses on the chemical design and the use of Surface-Enhanced Raman Scattering (SERS)-active nanotags for measuring surface markers that can be overexpressed at the surface of single cancer cells. Indeed, providing analytical tools with true single-cell measurements capabilities is capital, especially since cancer research is increasingly leaning toward single-cell analysis, either to guide treatment decisions or to understand complex tumor behaviour including the single-cell heterogeneity and the appearance of treatment resistance. Over the past two decades, SERS nanotags have triggered significant interest in the scientific community owing their advantages over fluorescent tags, mainly because SERS nanotags resist photobleaching and exhibit sharper signal bands, which reduces possible spectral overlap and enables the discrimination between the SERS signals and the autofluorescence background from the sample itself. The extensive efforts invested in harnessing SERS nanotags for biomedical purposes, particularly in cancer research, highlight their potential as the next generation of optical labels for single-cell studies. The review unfolds in two main parts. The first part focuses on the structure of SERS nanotags, detailing their chemical composition and the role of each building block of the tags. The second part explores applications in measuring overexpressed surface markers on single-cells. The latter encompasses studies using single nanotags, multiplexed measurements, quantitative information extraction, monitoring treatment responses, and integrating phenotype measurements with SERS nanotags on single cells isolated from complex biological matrices. This comprehensive review anticipates SERS nanotags to persist as a pivotal technology in advancing single-cell analytical methods, particularly in the context of cancer research and personalized medicine.

SERS nanotags for folate receptor α detection at the single cell level: discrimination of overexpressing cells and potential for live cell applications.

Folate receptor α (FRα) is a high affinity folate membrane receptor that is overexpressed in a wide variety of cancers. Detecting the overexpression of this receptor is important for cancer cells identification and to potentially guide the choice of treatment since several FRα-targeted drugs are currently in clinical trials. In this work, we built SERS nanotags based on core@shell Au@Ag nanoparticles labelled with resonant Raman-reporter and functionalised with a thiolated PEG linker bearing folic acid at the chain end. Using SERS mapping on single cells, we showed that the nanotags (FR-nanotags) could specifically target FRα on overexpressing HeLa cells and could measure the gradual blocking of FRα by free folic acid introduced in the media along the nanotags. With a control nanotag, we showed that the SERS response was 10-fold higher on HeLa cells when folic acid is present on the PEG linker compared to PEG chains without folic acid. Non-specific binding of the FR-nanotags was demonstrated to be low and mainly caused by the folic acid molecule at the PEG chain end. When comparing cancer cells with different expression levels of FRα, we obtained 4-fold higher SERS response on overexpressing HeLa cells compared to non-overexpressing A549 cells, allowing the discrimination of both cell lines with a high contrast. Owing to the biocompatibility of the developed nanotags, we demonstrated that measurements of FRα on live HeLa cells were also possible and gave similar results to measurements on fixed cells, indicating the versatility of the developed nanotags for detecting FRα under various experimental conditions.

Surface-assisted laser desorption/ionization mass spectrometry imaging: A review.

In the last decades, surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has attracted increasing interest due to its unique capabilities, achievable through the nanostructured substrates used to promote the analyte desorption/ionization. While the most widely recognized asset of SALDI-MS is the untargeted analysis of small molecules, this technique also offers the possibility of targeted approaches. In particular, the implementation of SALDI-MS imaging (SALDI-MSI), which is the focus of this review, opens up new opportunities. After a brief discussion of the nomenclature and the fundamental mechanisms associated with this technique, which are still highly controversial, the analytical strategies to perform SALDI-MSI are extensively discussed. Emphasis is placed on the sample preparation but also on the selection of the nanosubstrate (in terms of chemical composition and morphology) as well as its functionalization possibilities for the selective analysis of specific compounds in targeted approaches. Subsequently, some selected applications of SALDI-MSI in various fields (i.e., biomedical, biological, environmental, and forensic) are presented. The strengths and the remaining limitations of SALDI-MSI are finally summarized in the conclusion and some perspectives of this technique, which has a bright future, are proposed in this section.

Spatially resolved determination of the abundance of the HER2 marker in microscopic breast tumors using targeted SERS imaging.

Highly selective nanoprobes have been developed based on SERS-active Au@Ag nanoparticles protected by a PEG coating and functionalized with monoclonal antibodies against human epidermal growth factor receptor 2 (HER2). The PEG coating allows to drastically reduce unspecific interactions during incubation on tissues, while the monoclonal antibodies allow a highly specific targeting of HER2. Using the designed SERS nanoprobes combined with a spectral imaging and data weighting approach, we demonstrate the proportionality between the SERS signal and the amount of HER2 antigen on the cell membranes as measured by digital image analysis of IHC staining in microscopic breast tumors (linear fit R2 = 0.87). We also show that the level of expression of HER2 measured by SERS is significantly different between several microscopic tumor parts of the same tissue slide. Therefore, SERS is proving to be a suitable technique for the localized quantitative measurement of specific markers in breast cancerous tissues. Owing to its high multiplexing capabilities, SERS could be a future tool of choice for characterizing the molecular heterogeneity of tumors at the microscopic scale.

Dual-polarity SALDI FT-ICR MS imaging and Kendrick mass defect data filtering for lipid analysis.

Lipids are biomolecules of crucial importance involved in critical biological functions. Yet, lipid content determination using mass spectrometry is still challenging due to their rich structural diversity. Preferential ionisation of the different lipid species in the positive or negative polarity is common, especially when using soft ionisation mass spectrometry techniques. Here, we demonstrate the potency of a dual-polarity approach using surface-assisted laser desorption/ionisation coupled to Fourier transform-ion cyclotron resonance (SALDI FT-ICR) mass spectrometry imaging (MSI) combined with Kendrick mass defect data filtering to (i) identify the lipids detected in both polarities from the same tissue section and (ii) show the complementarity of the dual-polarity data, both regarding the lipid coverage and the spatial distributions of the various lipids. For this purpose, we imaged the same mouse brain section in the positive and negative ionisation modes, on alternate pixels, in a SALDI FT-ICR MS imaging approach using gold nanoparticles (AuNPs) as dual-polarity nanosubstrates. Our study demonstrates, for the first time, the feasibility of (i) a dual-polarity SALDI-MSI approach on the same tissue section, (ii) using AuNPs as nanosubstrates combined with a FT-ICR mass analyser and (iii) the Kendrick mass defect data filtering applied to SALDI-MSI data. In particular, we show the complementarity in the lipids detected both in a given ionisation mode and in the two different ionisation modes. Graphical abstract.

Multiplex micro-SERS imaging of cancer-related markers in cells and tissues using poly(allylamine)-coated Au@Ag nanoprobes.

Surface-enhanced Raman scattering (SERS) nanoprobes based on Au@Ag core@shell nanoparticles coated with poly(allylamine) were functionalized with small targeting molecules to evaluate simultaneously the level of expression of two cancer-related markers, both in cells and in tissues. The Au@Ag nanoparticles provide a high SERS signal enhancement in the visible range when combined with resonant Raman-active molecules. The poly(allylamine) coating plays a dual key role in (i) protecting the metal surface against the complex biological medium, leading to a stable signal of the Raman-active molecules, and (ii) enabling specific biofunctionalization through its amine functions. Using small targeting molecules linked to the polymer coating, two different nanoprobes (duplex approach) were designed. Each was able to specifically target a particular cancer-related marker: folate receptors (FRs) and sialic acid (SA). We demonstrate that the level of expression of these targeted markers can be evaluated following the SERS signal of the probes incubated on cells or tissues. The potential overexpression of folate receptors and of sialic acid was evaluated and measured in breast and ovarian cancerous tissue sections. In addition, FR and/or SA overexpression in the tumor region can be visualized with high contrast with respect to the healthy region and with high spatial accuracy consistent with histology by SERS imaging of the nanoprobe signal. Owing to the unique spectral signature of the designed nanoprobes, this approach offers an efficient tool for the spatially resolved, in situ measurement of the expression level of several cancer-related markers in tumors at the same time.Graphical abstract.