Engineering of plasmonic nanostructures for surface-enhanced Raman spectroscopic applications in liquid biopsy analysis
Diagnosis of cancer at early stages can significantly increase a patient’s survival rate and improve their quality of life. Tissue biopsy, considered as the clinically validated gold standard approach, has been applied to diagnose diseases and cancers. However, tissue biopsy faces a series of issues for wide practical utilizations, such as its invasive nature with associated risk and the difficulty of repeated sampling. Recently, liquid biopsy as an alternative candidate for clinical applications (e.g., early diagnosis) has been increasingly valued because it offers a non-invasive cancer screening option without having to obtain tumor tissue. It can be utilized in real-time to characterize the genetic landscape of cancerous lesions by analyzing specific disease-associated biomarkers from body fluids. Extensively studied biomarkers include circulating tumor-derived cells (CTCs), extracellular vesicles (EVs), circulating tumor nucleic acids (ctNAs, e.g., ctDNA, ctRNA) and proteins (e.g., cytokines). Nevertheless, the low levels of these circulating biomarkers against the background of plentiful non-targeted biomaterials pose technical challenges to traditional tools for accurate characterization and quantification. Therefore, there is an urgent demand to develop highly sensitive and specific approaches for probing the biomarkers of interest in a simple and rapid manner.
The overall aim of this thesis is to utilize surface-enhanced Raman scattering (SERS) spectroscopy as the main technique for detecting liquid biopsy biomarkers. The superior features of SERS include ultrasensitivity, high specificity, multiplexing capability, accessibility of molecular structure information, and photostability, which render it an ideal candidate for trace analysis and multi-component profiling.
Herein, to achieve highly sensitive and reliable SERS responses, the effort was first put into engineering novel nanostructures with controlled size, morphology, composition and surface charge, and exploring how these features influence SERS signal quality. Four types of nanostructures were rationally designed, synthesized and characterized by transmission electron microscopy (TEM), dynamic light scattering, UV-visible spectroscopy (UV-vis) and SERS. The optimal positively-charged nanostar with the highest SERS enhancement was considered the appropriate substrate for the analysis of negatively-charged targets.
We used positive nanostars to establish an analysis platform for intrinsically screening negatively charged small DNAs (e.g., the mutant BRAF V600E gene type). By integrating direct SERS measurement with polymerase chain reaction (PCR) and a statistical analysis method (principal component analysis-linear discriminate analysis, PCA-LDA), the mutant V600E gene was detected and discriminated from BRAF wild type (WT) gene in both cell line and human plasma samples. This demonstrates that a PCR/direct SERS strategy is capable to ultrasensitively and accurately detect DNA mutations in clinical application.
Crossing from a single biomolecule (a mutant DNA) to a macromolecule assembly, we applied the direct SERS strategy to analyze negatively-charged tumor-derived EVs. EVs represent a snapshot of the biocomponents of their parental cells. A combination of spectroscopic methods, including SERS, UV-vis, fluorescence excitation/emission and atomic force microscope-infrared spectroscopy (AFM-IR), was employed to characterize cancerous EVs. Their molecular profile based on different experimental variables (e.g., EV origin, isolation approach, and isolation batch) was explored via SERS and PCA-LDA discrimination. This assay delivers important information on EV spectroscopic profiling and arouses concerns on corresponding variations generated from different experimental settings.
As a direct SERS strategy cannot specifically aim at a target analyte and requires sample pre-treatment, we developed a specific indirect SERS approach by means of an antibody specific targeted towards a target molecule. We applied this approach to evaluate two disease-associated cytokines. This analysis was performed on a multi-layered fibrous polymer membrane using anionic, citrate-coated, spherical gold nanoparticle as the SERS substrate. This duplex sensing assay exhibited high sensitivity, great specificity and low cross-activity when two target cytokines were concurrently detected in phosphate-buffered saline and human serum, indicating its promising analysis capability for clinical samples.
One of the issues with nanoparticles in assays is their poor stability. We, therefore, encapsulated the negatively charged nanostars within a protective silica shell to improve their stability and functionalization capability. The synthesis and silica-encapsulation were optimized for better stability in high ionic strength environment. The silica encapsulated nanostars were then used as SERS nanotags to detect cancerous EVs. Their improved stability compared to the bare nanostars implies that the silica shell coating protects nanostars from the environmental triggers and the coated nanostars are qualified to be used to analyze liquid biopsy biomarkers from a complex biological sample.
In summary, this thesis re orts the development and application of a series of nano article SERS-based strategies to analyze biomarkers in liquid bio sies. These strategies offer more choices and information for real clinical application with desirable sensitivity and specificity. Surface charge-tunable nanostars have been found to play a critical role in SERS enhancement and its encapsulation within a silica shell achieved better stability for potential clinical applications. The proof-of-concept demonstrations of this thesis open the way to developing clinically relevant nanomaterial-based assay systems for circulating biomarkers analysis in liquid biopsy.