A CNT Switch for Identifying and Classifying Breast Cancer Tumor Cells in Plasma: Enhancing Selectivity of Molecular Assay with Nanoscale Hydrodynamic Shear

The goal of this research is to develop an inexpensive and non-invasive alternative to MRI scans and tissue biopsies currently used to detect metastatic breast cancer and its relapse. It is well established that Circulating Tumor Cells (CTCs) in blood stream plays a crucial role in determining metastases, and quantification of receptors ERBB2 (HER2), estrogen (ER) and progesterone (PR) within the CTCs provide valuable information regarding the stage of the cancer, treatment options and patient survival rates. Therefore, we report a highly sensitive and selective CNT-switch liquid biopsy platform to detect and monitor early metastasis and its relapse by analyzing protein expressions within the CTCs from the blood of breast cancer patients.

Detection and quantification of low-concentration proteins on circulating tumor cells are generally plagued by two distinct obstacles: lack of sensitivity due to high dissociation equilibrium constant KD; non-specificity due to an abundance of non-targets with similar KD. The detection sensitivity for our protein sensing platform is achieved with complete association of the antibody-antigen-antibody (Ab-Ag-Ab) complex by precisely and rapidly assembling carbon nanotubes (CNTs) across two parallel electrodes via sequential DC electrophoresis and dielectrophoresis (DEP), and with single-CNT electron tunneling conductance. The high selectivity is achieved with a critical hydrodynamic shear rate between the activated dissociation shear rates of target and non- target linkers of the aligned CNTs.

We are able to reach detection limits of 10 femtomolar (fM) for three breast cancer biomarkers homogeneous samples with high dissociation constants: HER2/HER2 antibody (0.44±0.07 nM), ER (0.35 nM) and PR (1 nM). We also demonstrate in spiked serum sample high selectivity towards target HER2 proteins against non-target HER2 isoform of a similar KD. The detection limit for HER2 in serum is lower than 100 fM. For all tests, irreversible capture and shearing allow us to tune the dynamic range up to 5 decades by increasing the CNT numbers.1 With three different breast cancer tumor cell line samples, we further validate the CNT-switch nanosensor platform’s capability to detect HER2, ER and PR protein expressions down to a single tumor cell. The quantifications of protein expressions per tumor cell allow us to develop cut-off r score values for breast cancer subtype classification. At last, the detection limit of the CNT- switch liquid biopsy platform for tumor cells spiked blood sample is 5 tumor cells within 7.5 ml of blood. This demonstrates the commercialization potential of the CNT-switch nanosensor as an early screening liquid biopsy technology for metastatic breast cancer and its relapse.