Monolayer Sensitivity Enables a 2D IR Spectroscopic Immuno-biosensor for Studying Protein Structures: Application to Amyloid Polymorphs

Abstract

Immunosensors use antibodies to detect and quantify biomarkers of disease, though the sensors often lack structural information. We create a surface-sensitive two-dimensional infrared (2D IR) spectroscopic immunosensor for studying protein structures. We tether antibodies to a plasmonic surface, flow over a solution of amyloid proteins, and measure the 2D IR spectra. The 2D IR spectra provide a global assessment of antigen structure, and isotopically labeled proteins give residue-specific structural information. We report the 2D IR spectra of fibrils and monomers using a polyclonal antibody that targets human islet amyloid polypeptide (hIAPP). We observe two fibrillar polymorphs differing in their structure at the G24 residue, which supports the hypothesis that hIAPP polymorphs form from a common oligomeric intermediate. This work provides insight into the structure of hIAPP, establishes a new method for studying protein structures using 2D IR spectroscopy, and creates a spectroscopic immunoassay applicable for studying a wide range of biomarkers.

Figure 1.

Schematic showing the workflow for the immunochemistry. (a) The gold is soaked with the antibodies and carbon disulfide in PBS buffer for about 24 h. (b) After antibodies are bound to the gold surface, they are rinsed in PBS buffer. (c) A 1% casein solution is then introduced to the flow cell to block any free binding sites. The blocking agents prevent nonspecific adsorption of any proteins, as confirmed in the experiments. At this point, a background transmission 2D IR spectrum is collected. (d) Finally, the hIAPP solution to be analyzed is incubated in the flow cell for 1 h before the sample is washed at least three times with PBS buffer.

Figure 2.

(a) 2D IR spectrum of the antibodies and casein on the gold surface. (b) 2D IR spectrum of the same sensor, but after addition and rinsing of the isotope-labeled amylin solution. (c) Normalized diagonal slices of the 2D spectra shown in panels a and b. (d) 2D IR difference spectrum generated by subtracting panel a from b. (e) Solution 2D IR spectrum of the same amyloid fibrils that bound to the antibody in panel d.

Figure 3.

(a) Bulk 2D IR spectrum of the G24 label in the aggregated state. (b) Difference spectrum of G24-labeled hIAPP in the isotope-labeled region of the amide-I spectrum. The spectrum is normalized to the most intense diagonal feature. (c) Interpolated diagonal slices of the spectra in panels a and b normalized to the main isotope feature in each spectrum.

Figure 4.

2D IR difference spectrum of monomeric hIAPP bound to the immunosensor.