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  • Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays.

Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays.

Nanotechnology (2016-02-24)
Farhad Khosravi, Patrick Trainor, Shesh N Rai, Goetz Kloecker, Eric Wickstrom, Balaji Panchapakesan
ABSTRACT

We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
Sigma-Aldrich
Mouse Anti-Human IgG, Fc Fragment Specific (HP6043) Peroxidase Conjugate, liquid, clone HP6043, Calbiochem®
Sigma-Aldrich
Anti-EpCAM-1 (Ab-1) Mouse mAb (VU-1D9), liquid, clone VU-1D9, Calbiochem®
Sigma-Aldrich
Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basis