Anastasiia Tukova

This work demonstrates the continuous-flow separation of ≈50 nm metal nanoparticles by their optical properties using counter-propagating laser beams. Metal nanoparticles are widely used, particularly in biotechnology, where their optical properties depend on size, shape, and chemistry. Heterogeneous mixtures are common, and greater homogeneity improves product performance and expands applications. We characterize the motion of gold and silver nanoparticles in solution under intense blue and green laser irradiation, imaging them via light scattering. When mixed and illuminated, 58 nm silver nanoparticles are primarily pushed by the blue laser, while 47 nm gold nanoparticles respond to the green laser. Continuous flow mitigates bulk temperature rise. This approach offers a potentially practical and efficient method for separating nanoparticles with desirable optical properties from less valuable byproducts, addressing a key challenge in nanoparticle synthesis.

DNA methylation, particularly the 5-methylcytosine modification, is the most prominent epigenetic modulation as it controls the expression of numerous genes in both healthy and diseased individuals. This has inspired the development of multiple diagnosis methods using DNA methylation as a cancer biomarker. However, DNA methylation analysis has not yet been established for routine cancer diagnosis due to the lack of clinical validation. Well-known methods, such as methylation-specific polymerase chain reaction (PCR), restriction enzyme-based digestion, and affinity-based enrichment, have not yet been standardized and often vary in their accuracy, sensitivity, and specificity from laboratory to laboratory. Surface-enhanced Raman scattering (SERS) has recently emerged as an alternative tool for methylation detection as it directly identifies DNA modifications through an intrinsic vibrational fingerprint, with the potential to reach single-molecule sensitivity for specific analytes under optimized conditions. Smart nanomaterial design enables orders-of-magnitude signal enhancement by optimizing probe-substrate interactions and advancing the substrate architecture. Although high-level summaries of various optical methods and comprehensive reviews of electrochemical methods have been published, this review provides, for the first time, an in-depth overview of SERS-based approaches for detecting cancer-specific DNA methylation, including their data analysis methods and potential clinical applications. The article emphasizes why SERS should be considered over existing methods as an emerging technique in DNA methylation analysis.

Methylation-specific PCR has been combined with a reporter-labeled SERS assay to detect gene-specific DNA methylation in the hMLH1 promoter region of the colorectal cancer genome.

Small extracellular vesicles (sEVs) are lipid bilayer-bound vesicles that carry critical biomarkers for disease detection. However, the inherent heterogeneity and complexity of sEV molecular characteristics pose significant challenges for accurate and comprehensive molecular profiling. Traditional analytical methods, including immunoblotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, exhibit several limitations, such as being time-consuming, requiring large sample volumes, and demonstrating relatively low sensitivity. Therefore, there is an urgent need to develop a highly sensitive and specific assay for the reliable detection of sEVs. Surface-enhanced Raman scattering (SERS) assays have emerged as a promising approach for sEV detection, offering advantages including high sensitivity and specificity. In the proposed SERS assay, SERS nanotags – comprising nanoparticles coated with Raman-active molecules and conjugated with antibodies – are employed to label surface-bound molecules on sEVs. This approach facilitates the generation of a high-intensity signal from molecules present in low abundance. Recently, anisotropic nanoparticles, such as star-shaped nanostructures, have garnered interest due to their ability to significantly amplify generated SERS signals for ultra-sensitive biomarker detection. In this study, we explore the application of gold nanostars (AuNSs) as SERS nanotags for the detection of sEVs. In principle, AuNS-based SERS nanotags were used to label the EpCAM protein, which can be found on the surface of cancer cell derived sEVs, and then sEV labelled SERS nanotags were captured by CD9-conjugated magnetic beads to form an immunocomplex, which exhibits a SERS signal. Our results demonstrate that the proposed SERS assay utilizing AuNSs provides high specificity and sensitivity, with a detection limit as low as 2.47 × 10³ particles per μL. Furthermore, the assay was tested with spiked plasma samples (cancer cell-derived sEVs spiked into healthy plasma), showing that its specificity remains unaffected by the presence of plasma. These findings suggest that the SERS assay incorporating AuNSs holds significant promise as an effective and reliable detection method for potential clinical applications.

DNA methylation has been widely studied with the goal of correlating the genome profiles of various diseases with epigenetic mechanisms. Multiple approaches have been developed that employ extensive steps, such as bisulfite treatments, polymerase chain reactions (PCR), restriction digestion, sequencing, mass analysis, etc., to identify DNA methylation. In this article, we report a facile label-free surface-enhanced Raman scattering (SERS) spectroscopy system that utilizes gold nanoparticles (AuNPs) for signal enhancement of methylated DNA. The key innovation of this work is to use anionic nanoparticles at a high ionic strength to introduce the aggregation of AuNPs with anionic DNA. When target methylated DNA is present, the presence of a methyl group in the cytosine C5 position of CpG sites induces a Raman peak at 1350 cm⁻¹. Our amplification-free system has a limit of detection (LOD) of 5% and a limit of quantification (LOQ) of 16% with good specificity. The method was applied to determine the hypermethylated levels of the germline of colorectal cancer cell lines SW48 and SW480. Our simple label-free method holds the potential to read the disease-associated methylation of genomic DNA.

Gold nanostars with 4-mercaptobenzoic acid as SERS nanotags show high biostability in cell culture media, with/without BSA precoating. SERS imaging reveals higher uptake by immune vs cancer cells; with protein precoating showing little effect.

Nonspherical gold nanoparticles (GNPs) are increasingly used to enhance sensitivity and selectivity in analytical methods such as surface-enhanced Raman spectroscopy (SERS) for detecting trace biomarkers. However, there is limited research on the adsorption properties of aromatic thiols onto gold nanoparticles of different morphologies, where surface curvature varies significantly at the molecular level. In this study, we investigated the adsorption kinetics of 4-mercaptobenzoic acid, an aromatic molecule, on GNPs with different shapes using SERS. Our findings revealed significant differences in the adsorption behavior and binding site preferences of aromatic thiols on GNPs with distinct morphologies. While thiol molecules consider any surface site on nanospheres equally appealing, nanostars exhibit variations in curvature and surface energy, leading to initial binding with further repositioning from the tips of the nanostar after plasmon activation. To address these differences, we proposed a universal method to evaluate the quantity of tightly bound adsorbed molecules on GNPs independently of the particle size, shape, or concentration.

Cancer-derived small extracellular vesicles (sEVs) are specific subgroups of lipid bilayer vesicles secreted from cancer cells to the extracellular environment. They carry distinct biomolecules (e.g., proteins, lipids and nucleic acids) from their parent cancer cells. Therefore, the analysis of cancer-derived sEVs can provide valuable information for cancer diagnosis. However, the use of cancer-derived sEVs in clinics is still limited due to their small size, low amounts in circulating fluids, and heterogeneous molecular features, making their isolation and analysis challenging. Recently, microfluidic technology has gained great attention for its ability to isolate sEVs in minimal volume. In addition, microfluidics allows the isolation and detection of sEVs to be integrated into a single device, offering new opportunities for clinical application. Among various detection techniques, surface-enhanced Raman scattering (SERS) has emerged as a promising candidate for integrating with microfluidic devices due to its ultra-sensitivity, stability, rapid readout, and multiplexing capability. In this tutorial review, we start with the design of microfluidics devices for isolation of sEVs and introduce the key factors to be considered for the design, and then discuss the integration of SERS and microfluidic devices by providing descriptive examples of the currently developed platforms. Lastly, we discuss the current limitations and provide our insights for utilising integrated SERS-microfluidics to isolate and analyse cancer-derived sEVs in clinical settings.