Quantitative ratiometric discrimination between noncancerous and cancerous prostate cells based on neuropilin-1 overexpression

Abstract

A multiplexed, ratiometric method is described that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro. The technique is based on bright surfaceenhanced resonance Raman scattering (SERRS) biotags (SBTs) infused with unique Raman reporter molecules, and carrying cellspecific peptides. Two sets of SBTs were used. One targets the neuropilin-1 (NRP-1) receptors of cancer cells through the RPARPAR peptide. The other functions as a positive control (PC) and binds to both noncancerous and cancer cells through the HIV-derived TAT peptide. Point-by-point 2D Raman maps of the spatial distribution of the two tags were constructed with subcellular resolution from cells simultaneously incubated with the two sets of SBTs. Averaging the SERRS signal over a given cell yielded an NRP/PC ratio from which a robust quantitative measure of the overexpression of the NRP-1 by the cancer cell line was extracted. The use of a local, on-cell reference produces quantitative, statistically robust measures of overexpression independent of such sources of uncertainty as variations in the location of the focal plane, the local cell concentration, and turbidity.surface-enhanced Raman spectroscopy biomarker | cancer cell identification | multiplexing | silver nanoparticles E arly and rapid identification of malignant cells [ideally freeflowing in biological fluids such as urine (1) or blood (2-4)] is a central goal in cancer research. Accordingly, seeking improvements in sensitivity and accuracy of detection and quantification through, for example, the development of cell-specific diagnostic tools and biomarkers is an active enterprise. Although it is known that such cells are present in body fluids, concentrating them to levels appropriate for confident identification and quantification is still a serious challenge, despite significant advances that have been made by, for example, using microfluidics (5). Once collected, the cells must be identified as either cancerous or noncancerous within tolerable confidence limits, which may be improved by, for example, using several nonredundant biomarkers. Numerous promising identification methods have been reported using antibodies. In this article, we report a technique based on surface-enhanced Raman spectroscopy (SERS) and biotags that produce bright surface-enhanced resonance Raman scattering (SERRS) signals, comparable to or exceeding the intensities of fluorescent tags. The SERRS biotags (SBTs) used in this study exploit peptides as recognition moieties. We show that using SBTs ratiometrically can provide highly statistically significant, quantitative measures of neuropilin-1 (NRP-1) overexpression insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, and turbidity.Normal Raman signals from cells and their infrared (FTIR) absorbance patterns have been previously used to differentiate between cancer and normal cells (6-9). SERS has been used as an alternative immunohistochemistry (IH...

Cancer characterization and diagnosis with SERS-encoded particles

“…In fact, it has been demonstrated that SEPs can adhere to tumor tissues in less than minutes, although the nonspecifically adsorbed SEPs must be removed by washing the tissue with serum (Wang et al 2014a). The obstacle set by the high background distribution of non-specifically bound nanoparticles can be overcome by implementing ratiometric approaches where one of the SEP type in the particle cocktail is used as a nonspecific internal reference to visually enhance the preferential adhesion of other targeting nanoparticles on tumor tissues (Mallia et al 2015;Oseledchyk et al 2017;Pallaoro et al 2011). Implementation of these methods is rather straightforward for SERS imaging due to the high degree of multiplexing provided by the narrow Raman linewidths.…”

“…The outer protective layer improves nanoparticle stability and prevents the desorption of the SERS codes from the particle surface. Further, the external stabilizing coating provides functional groups on their surface for further bioconjugation (e.g., antibodies or aptamers) for selective targeting Conde et al 2014;Pallaoro et al 2011).…”

Scanning angle Raman spectroscopy: Investigation of Raman scatter enhancement techniques for chemical analysis

“…Many of the strategies that have been developed to date use SERS substrates that contain a peptide or protein to target a specific cellular marker, often on the cell membrane, that is unique to cancer cells or has a differential expression in cancerous cells. 521,[548][549][550][551][552] Moskovits and coworkers developed one such strategy for distinguishing cancerous epithelium prostate cells from non-cancerous cells. 549 Their approach was to ratio the signal obtained from two separate Ag nanoparticle based SERS substrates to detect the over expression of neuropilin-1 in the cancerous cell line.…”

Direct Laser Synthesis of Ag Nanoparticles from Ammonia-alcoholic Solutions of AgNO3

“…Ag NPs have the most intensive plasmon absorption, 5 can be promising as a disinfecting addition in form of NPs' colloidal solutions, [6][7][8] can be used for the long-term control of the processes in cells of live organisms or as biomarkers in cancer diagnosis methods. 9 Chemical methods of NPs' synthesis (especially metallic ones) are well-known, [10][11][12] but, apart from NPs obtained as a result of reduction reactions or ion exchange, there are always by-products present, which confines the use of these methods. That is why lately in the last 10 years new alternative ways of getting metal NPs are being developed, in particular with the use of laser irradiation.…”