Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor for Label-Free Detection of Cancer Immunotherapy-Relevant Antibody-Ligand Interactions

Abstract

We fabricated a novel single molecule nanosensor by integrating a solid-state nanopore and a double nanohole nanoaperture. The nanosensor employs Self-Induced Back-Action (SIBA) for optical trapping and enables SIBA-Actuated Nanopore Electrophoresis (SANE) for concurrent acquisition of bimodal optical and electrical signatures of molecular interactions. This work describes how to fabricate and use the SANE sensor to quantify antibody-ligand interactions. We describe how to analyze the bimodal optical-electrical data to improve upon the discrimination of antibody and ligand versus bound complex compared to electrical measurements alone. Example results for specific interaction detection are described for 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. We also describe how to analyze the bimodal optical-electrical data to discriminate between specific and non-specific interactions between antibodies and ligands. Example results for non-specific interactions are shown for cancer-irrelevant TCRmAbs targeting the same pMHCs, as a control. These example results demonstrate the utility of the SANE sensor as a potential screening tool for ligand targets in cancer immunotherapy, though we believe that its potential uses are much broader.

Keywords: Antibody-ligand interactions; Dual modality nanosensing; Dual nanoholes; Nanopore translocations; Peptide major histocompatibility complexes (pMHCs); Plasmonic optical trapping; Solid-state nanopores; TCR-like monoclonal antibodies.

Fig. 1:

Optical lithography masks used for fabrication of SANE sensor. (a) Front side mask. (f) Backside mask. (b) & (g) Alignment markers to locate the side of the mask under an optical aligner. (c) Individual chip with specific markers (d), for backside alignment. (e) The 786 μm square window. (h) Individual chip in with specific markers (i), for front side alignment. (j) & (k) FIB alignment markers.

Fig. 2:

(a) Cross-section of the SANE sensor chip. (b) Image of wafer after patterning with back side mask and TMAH etching. (c) Image of wafer after patterning Au/Cr layer with the front side mask. (d) Image of individual diced chips ready for FIB milling after removal of sacrificial oxide layer.

Fig. 3:

Confocal microscope images of wafer at various stages of fabrication. (a) Image taken after etching away the silicon nitride from the patterned square window. Image taken after TMAH etching from (b) front side and (c) backside of the wafer. Image taken after patterning FIB alignment markers from (d) front side and (e) backside of the wafer. (f) Image taken after removal of sacrificial oxide layer. The white region or and vertical gap highlighted in the red rectangle box is a sign of undercut caused by etching of the backing SiO₂ layer.

Fig. 4:

(a) SEM micrograph of front side of the SANE chip before FIB drilling. (b) Design of the DNH structure. He ion microscope image of top view (c) and tilted view (d) of milled DNH with 17 % sidewall taper and a 25 nm ssNP drilled at its center.

Fig. 5:

(a) Schematic of the PDMS slab with the exact dimensions of the openings made into it. (b) Corresponding PDMS flow cell cross-sectional view with SANE sensor with dotted lines correlating to the regions of the openings in the PDMS slab. (c) Image of the PDMS slab bonded to a glass slide. (d) Attachment of the PDMS-glass slide flow cell onto the right-angle brackets. (e) Image of prepared PDMS flow cell with SANE chip. (f) Placement of flow cell on piezo-controlled stage.

Fig. 6:

(a) Image of prepared PDMS flow cell with SANE chip ready for placement on piezo-controlled stage. (b) PDMS flow cell cross-sectional view with SANE sensor. (c) Complete optical setup with PDMS flow cell placement and measurement instruments. LD: Laser Diode, QWP: Quarter Wave Plate, GTP: Glan-Thompson Polarizer, HWP: Half Wave Plate, 4x BE: 4x Beam Expander, MR: Mirror, OL: Carl-Zeiss 1.3 N.A. 63x Objective Lens, CL: Condenser Lens, PD: Photodiode. (d) Image of flow cell placed between objective and condenser lens.

Fig. 7:

(a) Plots of simultaneously recorded optical transmission (top, blue; V), raw ionic current (middle, red; nA) and 20 Hz low-pass filtered ionic current [bottom, green; (nA)] versus time (sec) for the single 20 nm silica nanoparticle trapped in the SANE sensor. Physical interpretation schematics for the signals recorded within gray-shaded regions A, B and C are shown in panels (b), (c) and (d), respectively. (b) Region A: Negatively charged nanoparticle entering the DNH-ssNP under applied bias. (c) Region B: Nanoparticle trapped and bobbing inside the DNH near the ssNP mouth. (d) Region C: Nanoparticle exiting the optical trap after the electrophoretic force dominates translocation. Data types measured from typical traces of (e) optical transmission and (f) ionic current.

Fig. 8:

Time traces of individual RAH antigen (a)-(c) and Anti-RAH antibody molecules (d)-(f). Optical transmission ((a) & (d)), raw ionic current ((b) & (e)) and filtered ionic current ((c) & (f)).

Fig. 9:

Comparison scatter plots of RAH-Antigen (300 nM) and Anti-RAH Antibody (300 nM) for all data types. (a) Electrical metrics alone. (b) Optical metrics alone. (c)-(d) Combined optical-electrical metrics.

Fig. 10:

Histogram of optical step change for RAH – Anti-RAH equimolar mixtures. A threshold of 4.20% (red dashed line) was defined for classifying events as bound complex (green region) versus unbound RAH (blue region) and Anti-RAH (yellow region).

Fig. 11:

(a)-(e) Optical and electrical time traces from an Anti-RAH equimolar mixture with RAH at 100 nM. (d)-(f) Corresponding time traces for an equimolar mixture of non-specific Anti-WNV antibody with RAH antigen at 100 nM.

Fig. 12:

Scatter plots of bimodal event metrics in specific (RAH – Anti-RAH, green circles, n = 95) versus non-specific (RAH – Anti-WNV, orange circles, n = 95) mixtures at 10 nM for all data types. (a) Electrical metrics. (b) Optical metrics. (c)-(d) Combined optical-electrical metrics.