Engineered nanopore of Phi29 DNA-packaging motor for real-time detection of single colon cancer specific antibody in serum

Abstract

The ingenious design of the bacterial virus phi29 DNA packaging nanomotor with an elegant and elaborate channel has inspired its application for single molecule detection of antigen/antibody interactions. The hub of this bacterial virus nanomotor is a truncated cone-shaped connector consisting of 12 protein subunits. These subunits form a ring with a central 3.6-nm channel acting as a path for dsDNA to enter during packaging and to exit during infection. The connector has been inserted into a lipid bilayer. Herein, we reengineered an Epithelial Cell Adhesion Molecule (EpCAM) peptide into the C-terminal of nanopore as a probe to specifically detect EpCAM antibody (Ab) in nanomolar concentration at the single molecule level. The binding of Abs sequentially to each peptide probe induced stepwise blocks in current. The distinctive current signatures enabled us to analyze the docking and undocking kinetics of Ab-probe interactions and determine the Kd. The signal of EpCAM antibody can be discriminated from the background events in the presence of nonspecific antibody or serum. Our results demonstrate the feasibility of generating a highly sensitive platform for detecting antibodies at extremely low concentrations in the presence of contaminants.

Figure 1

Illustration of the phi29 connector channel structure. (A) Structure of one subunit showing the location of the EpCAM probe (red). (B) Construction of the modified gp10 gene by insertion of His tag at the N-terminus, 6-Gly linker and EpCAM probe at the C-terminus. (C) top view; (D) side view and (E) section view of the phi29 connector showing the size of the channel and location of the EpCAM probe (red) and incorporated into the phi29 DNA packaging motor channel. (F) Molecular weight of wild type, C-His and N-His C-EpCAM connector on 10% SDS-PAGE. (G) Demonstration of the accessibility and specificity of EpCAM probe in the C-terminal of the phi29 connector channel by Far Western blot.

Figure 2

Characterization of membrane-embedded EpCAM engineered phi29 connector channels. (A) Continuous current trace showing multiple insertions of EpCAM reengineered connector channels into planar lipid bilayer. (B) Current voltage trace under a ramping voltage of −100 to 100 mV. (C) Conductance comparison of N-his C-EpCAM and C-his connector channel under the same buffer condition 75 mV.

Figure 3

Real time sensing of EpCAM antibody interactions with EpCAM engineered phi29 connector channels. (A) Before addition of EpCAM antibody. (B) Transient binding events. (C) Permanent binding events.

Figure 4

Analysis of current blockage induced by EpCAM probe/antibody interaction. (A) Histograms of transient current blockage. (B) Histograms of permanent current blockage events. (C) Scatter of dwell time versus current blockage.

Figure 5

Kinetic studies of EpCAM probe/antibody interaction based on transient dwell time events. (A) Event histograms of current time traces τ_on, which is the time between two consecutive binding events. (B) Event histograms of current time traces τ_off, which is the dwell time. (C) Frequency of association as a function of antibody concentration [P], which is linear fit to (1/τ⃗_on) = k_on×[P], yielding the association rate K_on. (D) Frequency of dissociation as a function of antibody concentration [P], which is a constant fit to (1/τ⃗_off) = k_off, yielding the dissociation rate K_off.

Figure 6

EpCAM antibody detection in the presence of high concentration of non-specific antibody and diluted serum. (A) Histogram of current blockage events caused by high concentration of nonspecific antibody. (B) Histogram of current blockage events caused by high concentration of non-specific antibody with EpCAM Ab. (C) Histogram of current blockage events caused by diluted serum. (D) Histogram of current blockage events caused by diluted serum with EpCAM Ab.