The use of plasmonic nanopores for single molecule detection has attracted considerable attention due to their high sensitivity and selectivity. In this study, we present a phase analysis approach for characterizing the trapping of single molecules in an AC-driven plasmonic nanopore. By analyzing the phase response of the plasmonic nanopore at select frequencies, we can differentiate between a test ligand, the antibody targeting this ligand, and the complexes that these ligands form, as well as observe their dynamics while inside the optical trap of the plasmonic nanopore. This pilot work shows the feasibility of a new approach for rapid and accurate identification of single molecules in complex mixtures.
Alternating current (AC) modulation of command voltage applied across a Self-induced Back Action Actuated Nanopore Electrophoresis (SANE) sensor, a type of plasmonic nanopore sensor that we have developed previously, enables acquisition of new data types that could potentially enhance the characterization of nanoparticles (NPs) and single molecules. In particular, AC voltage frequency response provides insight into the charge and dielectric constant of analytes that are normally obfuscated using DC command voltages. We first analyzed Axopatch 200B data to map the frequency response of the empty SANE sensor in terms of phase shift and amplitude modulation, with and without plasmonic excitation. We then tested the frequency response of 20 nm diameter silica NPs and 20 nm gold NPs trapped optically, which made these particles hover over an underlying 25 nm nanopore at the center of the SANE sensor. By applying a DC command voltage with a superimposed AC frequency sweep while keeping the NPs optically trapped in the vicinity of the nanopores’s entrance, we have found that silica and gold NPs to have distinctly different electrical responses. This pilot work demonstrates the feasibility of performing AC measurements with a plasmonic nanopore, which encourages us to pursue more detailed characterization studies with NPs and single molecules in future work.
A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses a Self- Induced Back-Action (SIBA) mechanism for optical trapping to enable SIBA-Actuated Nanopore Electrophoresis (SANE) sensing through a nanopore located immediately below the optical trap volume. The ligand-receptor model consisted of a Natural Killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A that was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand. The latter interaction pair was used as a simplified model of lowaffinity interactions between immune cells and peptide-presenting cancer cells that occur during cancer immunotherapy. A cancer-irrelevant GroEL ligand was also targeted by the same receptor in control experiments to test for non-specific interactions. Although the analysis of different pairs of bimodal SANE sensor signatures enabled some level of discrimination between specific and non-specific interactions the separation was not complete, which suggested the need for multi-dimensional data analyses in future work. Nevertheless, the SANE sensor showed ability to quantify the fast dissociation rate (koff) in this low-affinity model system that was previously shown to be challenging to quantify with commercial technologies. The koff value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE sensor as a screening tool in cancer immunotherapy.
Solid-state nanopores have gained much attention as a bioanalytical platform. By virtue of their tunable nanoscale dimensions, nanopore sensors can a spatial resolution that spans a wide range of biological species from a single-molecule to a single virus or microorganism. Several groups have already used solid-state nanopores for tag-free detection of viruses. However, no one has reported use of nanopores to capture a single virus for further interrogation by the electric field inside nanopores. In this paper we will report detection of single HIV-1 particle with solid-state nanopores and demonstrate the ability to trap a single HIV-1 particle on top of a nanopore and force it to squeeze through the pore using an electric field.
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