Effect of Varying Expression of EpCAM on the Efficiency of CTCs Detection by SERS-Based Immunomagnetic Optofluidic Device

SIMPLE SUMMARY: In this work we present a magnetically supported SERS-based immunoassay based on solid SERS-active support for the detection of circulating tumor cells. The SERS response in our optofluidic device was correlated with the level of EpCAM expression. The level of EpCAM cell expression in four cell lines with relatively high (human metastatic prostate adenocarcinoma cells (LNCaP)), medium (human metastatic prostate adenocarcinoma cells (LNCaP)), weak (human metastatic prostate adenocarcinoma cells (LNCaP)), and no EpCAM expressions (cervical cancer cells (HeLa) has been estimated using Western Blot method supported by immunochemistry and correlated with responses of immunomagnetic SERS-based analysis. The capture efficiency of developed assay was investigated in metastatic lung cancer patients. The assay demonstrates the capability to detect circulating tumor cells from blood samples over a broad linear range (from 1 to 100 cells/mL) reflecting clinically relevant amount of CTCs depending on the stage of metastasis, age, applied therapy. ABSTRACT: The circulating tumor cells (CTCs) isolation and characterization has a great potential for non-invasive biopsy. In the present research, the surface–enhanced Raman spectroscopy (SERS)-based assay utilizing magnetic nanoparticles and solid SERS-active support integrated in the external field assisted microfluidic device was designed for efficient isolation of CTCs from blood samples. Magnetic nanospheres (Fe(2)O(3)) were coated with SERS-active metal and then modified with p-mercaptobenzoic acid (p-MBA) which works simultaneously as a Raman reporter and linker to an antiepithelial-cell-adhesion-molecule (anti-EpCAM) antibodies. The newly developed laser-induced SERS-active silicon substrate with a very strong enhancement factor (up to 10(8)) and high stability and reproducibility provide the additional extra-enhancement in the sandwich plasmonic configuration of immune assay which finally leads to increase the efficiency of detection. The sensitive immune recognition of cancer cells is assisted by the introducing of the controllable external magnetic field into the microfluidic chip. Moreover, the integration of the SERS-active platform and p-MBA-labeled immuno-Ag@Fe(2)O(3) nanostructures with microfluidic device offers less sample and analytes demand, precise operation, increase reproducibly of spectral responses, and enables miniaturization and portability of the presented approach. In this work, we have also investigated the effect of varying expression of the EpCAM established by the Western Blot method supported by immunochemistry on the efficiency of CTCs’ detection with the developed SERS method. We used four target cancer cell lines with relatively high (human metastatic prostate adenocarcinoma cells (LNCaP)), medium (human metastatic prostate adenocarcinoma cells (LNCaP)), weak (human metastatic prostate adenocarcinoma cells (LNCaP)), and no EpCAM expressions (cervical cancer cells (HeLa)) to estimate the limits of detection based on constructed calibration curves. Finally, blood samples from lung cancer patients were used to validate the efficiency of the developed method in clinical trials.