Photonic crystal kinase biosensor

Abstract

We have developed a novel biosensor for kinases that is based on a kinase-responsive polymer hydrogel, which enables label-free screening of kinase activity via changes in optical properties. The hydrogel is specifically designed to swell reversibly upon phosphorylation of a target peptide, triggering a change in optical diffraction from a crystalline colloidal array of particles impregnated into the hydrogel. Diffraction measurements, and charge staining, confirmed the responsive nature of the hydrogel. Moreover, the change in diffraction of the hydrogel upon treatment with kinase exhibited a time- and dose-dependent response. A theoretical model for ionic polymer networks describes the observed optical response well and can be used to quantify the extent of phosphorylation.

Scheme 1. Fabrication of a Kinase Responsive CCA Biosensor

Figure 1

Red shift in peak optical reflectance of hydrogel-encapsulated CCAs with increasing concentration of immobilized negatively charged groups (at pH 5.5). The inset images (∼5 × 5 mm²) show visual color changes of the hydrogels. The total concentrations of negative charge from 5 to 25 mM, as indicated above each image, were measured by colorimetric staining.

Figure 2

Concentration of immobilized charge in the kinase responsive CCA-containing hydrogels characterized by colorimetric staining. The blue and red bars represent negative (−) and positive (+) charge concentrations, respectively. The positive charge concentration in the CCA-containing hydrogel and the hydrolyzed hydrogel were determined to be negligible within error. Error bars represent ±1σ, as measured for the first three steps for 30 samples. The PKA treatment was performed at a concentration of 16 U/μL and 8 h and is reported for 3 independent samples.

Figure 3

Red shift in the wavelength of peak diffraction as a function of (a) time upon treatment with 16 U/μL of PKA and (b) PKA concentration for 2 h treatments. The solid black curves represent model predictions over the range fitted, and the dashed curves are extrapolations from the model. Error bars represent ±1σ from the mean for 3 independent samples.

Figure 4

Sensitivity of kinase responsive CCA sensor to PKA inhibition by H-89 (inset structure). Inhibition of PKA by H-89 was measured with varying inhibitor concentrations (0–10⁵ nM) and 16 U/μL of PKA. Hydrogel-encapsulated CCAs were incubated with the enzyme and inhibitor for 3 h at 30 °C. Error bars represent ±1σ from the mean for 3 independent samples.