Nanohole-Based Surface Plasmon Resonance Instruments with Improved Spectral Resolution Quantify a Broad Range of Antibody-Ligand Binding Kinetics

Abstract

We demonstrate an affordable low-noise SPR instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10−6 without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 milliseconds followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools.

High-Affinity Binding of Remyelinating Natural Autoantibodies to Myelin-Mimicking Lipid Bilayers Revealed by Nanohole Surface Plasmon Resonance

“…However, in previous work, our group compared kinetic rate constants and K D values obtained from a nanohole SPR instrument to those obtained from a commercial SPR instrument. 16 Antibodies binding to non-lipid antigens with known K D values ranging from 200 pM to 40 nM were examined. For all binding interactions we studied, the rate constants and K D values obtained with nanohole SPR were quite similar to those measured with a commercial Biacore 3000 system.…”

Plasmon-enhanced optical sensors: a review

“…43,44 The plasmonic nano-hole array is particularly suitable for integration into microfluidics, 45 enabling real-time measurement of antibody-ligand binding kinetics 46 and/or multiplexed detection. 47 For example, Pang et al demonstrated a high-performance, microfluidic nano-hole array with a refractive index sensitivity of 1520 nm/RIU based on sequential injection of ethylene glycol in water.…”

Strategies for enhancing the sensitivity of plasmonic nanosensors

“…Additionally, the refractive index sensitivity of gold nanorods is linearly proportional to the aspect ratio, facilitating its tunability [97]. Nanostructures of other shapes, including nanocubes [106][107][108][109], nanoshells [110][111][112], nanodiscs [113][114][115], nanotriangles [116,117], nanostars [118][119][120], nanobipyramids [121,122], nanorice [123,124], nanoholes [125,126], and nanocrescents [56,[127][128][129][130], have also been investigated and expanded to nanocubes and nanocuboids enclosed with concave surfaces (Fig. 1).…”