We present a novel method to trap nanoparticles in double nanohole (DNH) nanoapertures integrated on top of solid-state nanopores (ssNP). The nanoparticles were propelled by an electrophoretic force from the cis towards the trans side of the nanopore but were trapped in the process when they reached the vicinity of the DNH-ssNP interface. The self-induced back action (SIBA) plasmonic force existing between the tips of the DNH opposed the electrophoretic force and enabled simultaneous optical and electrical sensing of a single nanoparticle for seconds. The novel SIBA actuated nanopore electrophoresis (SANE) sensor was fabricated using two-beam GFIS FIB. Firstly, Ne FIB milling was used to create the DNH features and was combined with end pointing to stop milling at the metal-dielectric interface. Subsequently, He FIB was used to drill a 25 nm nanopore through the center of the DNH. Proof of principle experiments to demonstrate the potential utility of the SANE sensor were performed with 20 nm silica and Au nanoparticles. The addition of optical trapping to electrical sensing extended translocation times by four orders of magnitude. The extended electrical measurement times revealed newly observed high frequency charge transients that were attributed to bobbing of the nanoparticle driven by the competing optical and electrical forces. Frequency analysis of this bobbing behavior hinted at the possibility of distinguishing single from multi-particle trapping events. We also discuss how SANE sensor measurement characteristics differ between silica and Au nanoparticles due to differences in their physical properties and how to estimate the charge around a nanoparticle. These measurements show promise for the SANE sensor as an enabling tool for selective detection of biomolecules and quantification of their interactions.
Recent advances in plasmonic nanopore technologies have enabled the use of concurrently acquired bimodal optical-electrical data for improved quantification of molecular interactions. This work presents the use of a new plasmonic nanosensor employing self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) for quantifying antibody-ligand interactions. T-cell receptor-like antibodies (TCRmAbs) engineered to target peptide-presenting major histocompatibility complex (pMHC) ligands, representing a model of target ligands presented on the surface of cancer cells, were used to test the SANE sensor's ability to identify specific antibody-ligand binding. Cancer-irrelevant TCRmAbs targeting the same pMHCs were also tested as a control. It was found that the sensor could provide bimodal molecular signatures that could differentiate between antibody, ligand and the complexes that they formed, as well as distinguish between specific and non-specific interactions. Furthermore, the results suggested an interesting phenomenon of increased antibody-ligand complex bound fraction detected by the SANE sensor compared to that expected for corresponding bulk solution concentrations. A possible physical mechanism and potential advantages for the sensor's ability to augment complex formation near its active sensing volume at concentrations lower than the free solution equilibrium binding constant (K D ) are discussed.
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 self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off 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 senor as a screening tool in cancer immunotherapy.
Efficient cancer cell capture has been previously demonstrated on functionalized surfaces with defined nanotopography such as nanoscale texture. However, conventional additive and subtractive methods to achieve nanotexture require access to specialized nanofabrication equipment within a dedicated cleanroom environment. Here, we present a technique to create flexible polydimethylsiloxane (PDMS) surfaces with high roughness (R q ∼680 nm) using a molding approach that can be performed in a standard laboratory environment. We also demonstrate a one-step integration of nanotextured PDMS into a reversibly sealed easy access modular microfluidic platform to simplify cell capture workflows (e.g. cell introduction, capture, and isolation). In our proof of concept, we characterize nanotextured PDMS surfaces and investigate the effect of increased surface area on cancer cell adhesion strength and capture compared to non-textured (plain) PDMS. We found that cells attached more strongly to antibody-functionalized nanotextured surfaces (26%±5% increase in threshold fluid shear stress, τ 50% ) and captured 71%±19% more cancer cells than functionalized plain surfaces. Our reversibly sealed microfluidic platform enabled a user-friendly method to access cells for post-capture analysis, and we report an efficient nucleic acid isolation process. We anticipate the easy fabrication, one-step microfluidic integration, and streamlined experimental workflow will simplify the incorporation of nanotextured substrates in applications that investigate cell-surface interactions.RECEIVED
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