Enhanced Raman spectroscopy as a tool for the improvement of cervical cancer screening: a comparison of global and targeted approaches

Half a million women worldwide are diagnosed with cervical cancer annually, with 87% of mortalities occurring in developing economies. HPV vaccination has reduced disease incidence significantly but must be supplemented with regular cervical screening. In established practice, cells from the cervix are fixed, stained and observed manually using an optical microscope. Inter-observer subjectivity and false diagnoses are a relatively frequent occurrence. The use of molecular pathology allows objective diagnoses to be made based on changes to the molecular content of cells and tissue with disease. This quantitative, objective and automated pathological analysis could be integrated with existing services, as well as creating potential for economically deprived regions. Molecular pathology through spectroscopic methods has shown great promise. For Raman spectroscopy, this is broadly categorised into global comparison or targeted biomarker approaches. In the global paradigm, a direct comparison of wavelength modulated with standard Raman spectroscopy showed marked improvement of sensitivity and specificity in fixed cell classification. Intracellular sampling location studies indicated that the technique is more robust for fixed than live cells. A functional nanoparticle bioconjugate for the established marker p16 was developed for targeted studies following optimisation of covalent, passive and bioconjugation methods. This was applied to fixed smear analogues, both lysed and intact, in lateral flow assay and SERS mapping contexts respectively. Considerations of hybrid SERS-SRS and global-targeted imaging were also explored, combining key vibrational modes and labelling moieties from both arms of the study. After consideration of the clinical context, the SERS-active paper-based dipstick assay was selected for further development. Novel binding peptides for p16 capture were discovered and validated, alternative consensus motifs found through next-generation sequencing and SERS-based quantification demonstrated to be sufficiently sensitive to detect relevant cellular analogues. The investigations described provide both a roadmap for further development of multiple Raman diagnostic tools in a clinical context and the foundational components of a point-of-care, inexpensive test for p16 which could streamline and broaden access to cervical screening