Surface-Enhanced Raman Scattering Tags for Rapid and Homogeneous Detection of Circulating Tumor Cells in the Presence of Human Whole Blood

Abstract

Surface-enhanced Raman scattering (SERS) is under active investigation in biomedical diagnostics due to its high sensitivity, increased levels of multiplexing, robustness, and ability to perform detection in blood and other biological matrices. SERS has been successfully applied for labeling cells 1 and tissues, 2 for multiplexed biomarker labeling to monitor apoptotic processes, 3 and for real-time monitoring of single live cell signaling processes. 4We have developed SERS tags (Nanoplex biotags, a trademark of Oxonica Inc.), comprised of one or more SERS-active metal nanoparticles (Au) and a sub-monolayer of reporter molecules adsorbed to the metal surface, all encapsulated in a protective and functionalized silica coat ( Figure 1A). 5 Nanoplex biotags have been successfully used in biological applications. 6,7 Here we describe a novel application of Nanoplex biotags for the direct detection of rare cancer cells in whole blood.Malignant cells are shed and circulate in the bloodstream of patients with solid tumors. 8 Since the "seed and soil" theory for circulating tumor cells (CTCs) was hypothesized 9 and confirmed, 10 two major approaches, based on polymerase chain reaction or cytometric methods (such as the CellSearch system), have been established for CTC detection. 8,11 However, high instrument cost and labor-intensive and time-consuming procedures remain a major concern and hamper their use in clinical diagnostics. Taking advantage of the intrinsic properties of the SERS tags, we have developed a novel, homogeneous, no-wash assay platform that overcomes the current assay limitations. We use magnetic beads for CTC capture and Nanoplex biotags for rapid and sensitive detection directly in human whole blood. Scheme 1 illustrates the concept in which magnetic beads, conjugated to an epithelial cell-specific antibody (epithelial cell adhesion molecule, anti-EpCAM), and the SERS tags, conjugated to an anti-her2 antibody (human epidermal growth factor receptor-2), bind to a tumor cell. Since the breast cancer cell is of epithelial origin, the magnetic bead-EpCAM antibody will specifically bind to this tumor cell but not regular circulating blood cells. Since the her2 receptor is highly expressed on the breast cancer cell membrane, the anti-her2-SERS tag will specifically recognize these tumor cells. By adding the magnetic bead-EpCAM and SERS tag-her2 conjugates to a patient's blood sample, circulating breast cancer cells (CTCs) can be detected rapidly and with good sensitivity in the presence of whole blood.In a proof-of-concept experiment, the breast cancer cell line SKBR3, expressing high levels of her2 receptor on the cell surface, 12 was used as a model target. After a 30 min incubation of SKBR3 cells with magnetic bead-EpCAM and SERS-her2 conjugates, small volumes of the reaction mixtures were loaded on a glass slide. Bright-field microscopy imaging showed specific binding of the magnetic beads-EpCAM to the tumor cells ( Figure 1B), and anti-her2

SERS labels for quantitative assays: application to the quantification of gold nanoparticles uptaken by macrophage cells

“…[4][5][6] Several bright examples about the application of SERS labels in nanobiotechnology and nanomedicine have been reported in recent years and various SERS labels have been developed. 18,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] For instance, gold nanoparticles capped with thiol terminated oligonucleotides composed of a Raman active molecule on the inner end and a DNA capture sequence have been used for the multiplexed detection of femtomolar amounts of more than six different DNA sequences in the same microarray. 20 In an analogous microarray set up, gold nanoparticles (AuNPs) with a targeting protein allowed the ultrasensible detection of proteins.…”

Surface enhanced Raman spectroscopy (SERS): Potential applications for disease detection and treatment

“…Ranging from metallic nanoparticles labelled with reporter molecules [65][66][67][68][69] to more complicated systems with mixed reporter monolayers [70], dually functionalised systems consisting of reporter molecules and membrane penetrating or targeting antibodies [71][72][73], peptides [65,74,75] or oligomers. To minimise or prohibit degradation from the surrounding environment nanostructures can be afforded a certain degree of stability by silica [13,76] or polymer encapsulation.…”

“…Unlike other applications, which might be concerned with the primary detection of cancer, CTCs are particularly aggressive and are associated with the initiation of further cancerous growth. [73,94] During an initial study the CTCs were exposed to antibody functionalised nanotags and magnetic beads. The nanotags whilst simultaneously binding to the CTCs acted as the reporter component and similarly, the magnetic beads whilst capable of binding to the cancer cells were incorporated so that the samples could be concentrated via a magnet.…”

Silica-coated dimers of silver nanospheres as surface-enhanced Raman scattering tags for imaging cancer cells

“…A common use of SERS for quantitative analysis is based on SERS tags [10][11][12][13][14], which can be fabricated by placing Raman reporter molecules on the surface of a metal nanoparticle (typically made of Ag or Au) to provide a simple way to code the surface property (e.g. the type of receptor or ligand) of tag with a known SERS spectrum.…”