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 nano-motor 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 twelve 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 nano-molar concentration at the single molecule level. The binding of Abs sequentially to each peptide probe induced step-wise 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 non-specific 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.

Binomial distribution for quantification of protein subunits in biological Nanoassemblies and functional nanomachines

“…11–13 The complexity and intricacies of these natural nanoparticles have inspired the development of biomimetic nanodevices. 14,15 These nanomachines or patterned structures can be manipulated to build sophisticated nanodevices or parts for arrays, chips, MEMS, 16 actuators, 17 molecular sensors, 18–20 molecular sorters, 21 single pore DNA sequencing apparatus 11–13 or other revolutionary electronic and optical devices 22,23 ex vivo . Medical applications of these or their derivatives could be used for pathogen detection, disease diagnosis, and drug and therapeutic RNA delivery.…”

“…Medical applications of these or their derivatives could be used for pathogen detection, disease diagnosis, and drug and therapeutic RNA delivery. 19,20,24,25 Also common to living system is the transportation of dsDNA. In the ASCE (additional strand catalytic E) family including the AAA+ (ATPases Associated with diverse cellular Activities) and the FtsK-HerA superfamily, there are nanomotors that facilitate a wide range of functions involved in dsDNA riding, tracking, packaging, and translocation.…”

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

“…The revolution mechanism offers a hint for the design of nanomolecular motors that can manipulate biomolecules in a controlled fashion. These nanomachines can also be applied for the construction of sophisticated nanodevices, including molecular sensors (221)(222)(223), bioreactors (224), chips (225), DNA-sequencing apparatuses (169,221,(226)(227)(228)(229), or other electronic and optical devices (230,231). In addition, these multisubunit biocomplexes could serve as drug targets for therapeutics and have potential for diagnostic applications (222,223,232,233), especially as the biomotors have an advantage of a high stoichiometry that could be applied to solve the drug resistance issue (234,235).…”

Selective Multidetection Using Nanopores

“…Advances in nanotechnology have enabled the development of nanogap nanopores as improved solid-state nanopores, which were initially created to address mechanical durability issues associated with bionanopores. These nanopore devices have been developed using different materials, 10 e.g., channel proteins for bionanopores 32,[55][56][57] , and graphene 53,58-79 and silicon semiconductor materials 80 for solidstate and nanogap nanopores. Emerging devices based on electric current changes, and not on tunneling currents, have also been proposed.…”