Integrated liquid biopsy for cancer diagnostics via genomic biomarkers detection

Cancer is one of the leading causes of mortality worldwide, accounting for about 10 million deaths annually. The survival rates can be improved by an early-stage diagnosis achieved by regular population screening, but existing methods are invasive (tissue biopsy), expose patients to radiation (image-based techniques), or show limited accuracy (blood and stool tests). Liquid biopsy is a promising minimally invasive alternative method based on the analysis of body fluids, like blood, saliva, or urine, for the presence of cancer-related biomarkers. Genomic biomarkers based on DNA and RNA alterations are of particular interest because of the contribution of genetic changes to cancer onset and progression. The currently used gold standard for nucleic acids detection, fluorescence-based techniques, requires a multi-step assay and labeling with a fluorescent dye due to weak auto-fluorescence from biological samples. This thesis aims to explore alternative methods with more straightforward assays and better multiplexing abilities for genomic biomarker detection. Surface-enhanced Raman Spectroscopy (SERS) has a promising potential for liquid biopsy application due to its ability to provide the qualitative and potentially quantitative composition of a sample by analyzing the enhanced inelastic scattering of light. An amplification of Raman scattering is achieved by metal NPs arranged as a colloidal solution, as an array on a solid substrate, or as a uniform nanostructure. The selective detection of DNAs and RNAs from body fluids requires biological protocols based on the application of DNAs complementary to the target. Chapter 1 provides details about the working principle of SERS and a literature review of the biological protocols for target-specific capturing. Chapter 2 presents the fabrication and testing of SERS substrates based on NPs colloidal solution and NPs on the solid substrate. Chapter 3 studies the application of one of the biological protocols for the SERS-based detection of microRNAs. Chapter 4 explores the application of photonic crystal fibers as a microfluidic device for the SERS-based measurement of microRNAs. Chapter 5 discusses the possibility of achieving spatially resolved chemical measurement with a technique similar to SERS called tip-enhanced Raman Spectroscopy. This thesis has shown that there is significant research attention towards the SERS-based detection of genomic biomarkers, especially the ones based on the dysregulation of microRNA expression level. The literature review has shown that some one-to-two-step biological protocols with relatively straightforward assays have a detection ability comparable to fluorescence-based sensing and even better multiplexing abilities. The SERS substrates developed during this PhD have shown the limit of detection of down to 10-8 M with a promising potential for further improvements. The use of photonic crystal fibers was found to be an exciting research direction due to the longer interaction area between the light, NPs, and the analytes, and further optimization of the experimental parameters can be achieved with the COMSOL model presented in the thesis. The last chapter also has shown that there are other techniques to amplify Raman scattering that can provide even more advantages, like spatially resolved measurements, and that also have a potential for liquid biopsy application.