(244h) A Shear-Enhanced CNT-DEP Nanosensor Platform for Single Cell Protein Assay

Breast cancer is the main cause of cancer deaths among women, with approximately 1.4 million new cases and nearly 0.5 million deaths worldwide per year. As the growth of tumor varies from patients to patients, its mortality rate can be reduced if the hormone receptor status of ER, PR and HER2 can be accurately determined from cells within tumors. Since tumor cells are heterogeneous, it is desirable to analyze individual cell rather than an average bulk measurement in order to extract accurate information. However, current high-throughput and quantitative technology Reverse Phase Protein Array (RPPA) still require at least 10 cells equivalents of sample ¹.Hence, there is a need to develop a detection platform for single-cell proteomics that will enable precise treatment plan based on individual’s specific information within tumor cells and significantly reduces the side effects of therapeutics.

We report a new nanoscale protein sensor platform that uses irreversible electrokinetic phenomena to achieve single cellular sensitivity and specificity for HER-2 protein assay. The sensitivity is achieved with irreversible rare events, driven by a DEP (dielectrophoresis) driven (antibody-antigen-antibody) Ab-Ag-Ab ELISA complex association, which can nevertheless be detected with a sensitive CNT electron tunneling conductance sensor design. The selectivity is achieved by working at a critical shear rate that is between the dissociation shear rate of the target and the non-target. Specific components of this new protein sensing platform include: 1. Association of protein targets with Ab probes functionalized onto an gold electrode pair; 2.Transportation and trapping of the second linking Ab probe functionalized CNTs with long-range DC electrophoresis and short-range AC DEP to the electrodes (lock); 3.Selective removal of assembled CNTs with non-targets by an optimized nanoshear protocol; 4.Conductance quantification of remaining bridging CNTs with target linkers (switch). Steps 1 and 2 involve rapidly driven dynamic events to prevent the captured targets from dissociating from the probe to reach the thermodynamic coverage. Optimized shear at the nm-high hydrodynamic slip length in step 3 irreversibly removes CNTs with non-targets due to the large Stokes drag of their high aspect-ratio cylindrical geometry. With only target-linked CNTs remaining, the digital conductance by electron-tunneling across the Ab-Ag-Ab complex allows us to reach a detection limit of 10,000 molecules (10 fM) for spiked HER2 samples. Irreversible capture and shearing also allow us to tune the dynamic range up to 100 billion (100 pM or 5 decades) by increasing the CNT number. The spiked sample results also demonstrated high selectivity towards target HER2 proteins against non-target HER2 isoform of a similar K_D. We will also carefully conduct experiments with real tumor cell lysate, and the number of proteins detected will be correlated with respect to tumor cell numbers.

References:

1. Neeley ES, Baggerly KA, Kornblau SM, Surface Adjustment of Reverse Phase Protein Arrays using Positive Control Spots, Cancer Inform., 2012; 11; 77-86.