Hydrogel gratings with patterned analyte responsive dyes for spectroscopic sensing

Abstract

This is an unprecedented report of hydrogel gratings with an analyte responsive dye immobilised in alternating strips where the patterned dye is its own dispersive element to perform spectroscopy. At each wavelength, the diffraction efficiency of hydrogel gratings is a function of dye absorbance, which in turn is dependent on the concentration of analytes in samples. Thus, changes in intensity of diffracted light of hydrogel gratings were measured for sensing of analytes. Equally, the ratio of diffracted intensities at two wavelengths was used for quantification of analytes to reduce errors caused by variations in intensity of light sources and photobleaching of dyes. 15.27 μm pitch gratings were fabricated by exposing 175 μm thick films of photofunctionalisable poly(acrylamide) hydrogel in a laser interferometric lithography setup, generating an array of alternating lines with and without free functional groups. The freed functional groups were reacted with pH sensitive fluorescein isothiocyanate to create gratings for measurement of pH. The ratio of intensity of diffracted light of hydrogel gratings at 430 and 475 nm was shown to be linear over 4 pH units, which compares favourably with ∼2 pH units for conventional absorption spectroscopy. This increased dynamic range was a result of cancellation of the opposite non-linearities in the pH response of the analyte responsive dye and the diffraction efficiency as a function of dye absorbance.

Fig. 1. (a) Laser IL set-up (α and β are apex and crossing angles, respectively) and (b) set-up for diffraction studies where inset shows the dimensions of the flow cell.

Fig. 2. Fluorescence microscope images and corresponding cross-sectional profiles of hydrogel films patterned using the IL set-up comprising FB with α of (a and c) 179 deg and (b and d) 177 deg.

Fig. 3. Diffraction patterns 40.13 μm pitch hydrogel gratings illuminated with lasers of peak wavelength of (a) 532 nm and (b) 650 nm.

Fig. 4. Gray scale value of first diffracted order versus wavelength when hydrogel gratings are irrigated with 10 mM phosphate buffer solutions of different pH where inset shows absorption spectrum of 5 μg ml⁻¹ fluorescein solutions of different pH in 1 cm pathlength cuvettes.

Fig. 5. Plots of (a) gray scale values of diffracted orders at three wavelengths and (b) ratio of gray scale values and absorbances at 430 and 475 nm for hydrogel gratings and conventional spectroscopy, respectively, as a function of pH of solutions (error bars are ±1 standard deviation derived from the intensity variations around the wavelengths of interest).

Fig. 6. Plot of gray scale difference around 430 and 475 nm as a function of time as the pH was alternated between 5 and 9.