A proof-of-principle study for performing enzyme bioassays using substrates immobilized in a leaky optical waveguide

This work is a proof-of-principle study on the feasibility of performing enzyme bioassays using a dye-doped leaky waveguide (DDLW) where the substrate was immobilised in the entire volume of the waveguide and the fraction of light confined in the waveguide interacted with the absorbing product formed as a result of the enzyme's action on the substrate. The immobilisation of the substrate in the waveguide offers the following benefits: (1) The coloured product was still immobilised in the waveguide, and thus unable to diffuse away from the sensing region in the waveguide, which would otherwise lead to a rapid loss of the absorbance signal. (2) The interaction between the optical mode and the immobilised product in the waveguide was maximised, resulting in an experimentally determined sensitivity 57.5 times higher than total internal reflection (TIR). (3) Because of the small volume of the waveguide, a high local concentration of ∼1.61 mM could be achieved using a small amount of substrate (7.29 pmol). This is ∼100 times lower than the case where the same concentration of the substrate solution is present in a microfluidic flow cell of typical dimensions. (4) The high local concentration of the substrate ensured that the rate of product formation was largely dependent on the concentration of the enzyme in the waveguide. This work demonstrated the suitability of DDLW to perform enzyme bioassays using fluorescein diacetate 5(6)-isothiocyanate and esterase, and the formation of fluorescein was monitored by recording changes in the intensity of the reflected light at the resonance angle. The DDLW has potential applications in drug discovery, clinical diagnostics and industrial biotechnology.