The waveguide layer of diffraction-based leaky waveguides (LWs) must be made of materials that have low refractive index, are permeable to analytes, can be deposited by spin coating, and can be functionalised and crosslinked. These requirements are fulfilled by thin films of chitosan hydrogels. In this work, we studied the reproducibility of diffraction-based LWs with chitosan waveguides. The average refractive index sensitivity (RIS) and RI limit of detection (LOD) of the eight devices investigated herein were 125.5 ± 3.8 deg RIU⁻¹ and 1.9 × 10⁻⁶ ± 1.3 × 10⁻⁶ RIU, respectively. While several challenges associated with the realisation of reproducible chitosan LWs have been addressed, reducing the variations in RI LOD requires improving the adhesion of chitosan films to glass substrates, minimising bubbles trapped in microfluidic channels, and using pumps with minimal pulsations. We showed that the buffer baseline of LWs with unmodified chitosan before and after introducing 750 μM bovine serum albumin (BSA), which is equal to the physiological levels of serum albumin, was different by 3.6%. Nevertheless, using biotin, anti-biotin antibody and BSA as exemplar recognition element, analyte and interferent, respectively, we demonstrated that diffraction-based chitosan LWs were suitable for monitoring analyte-RE binding in the presence of 750 μM BSA.

Broadband absorption spectroscopy integrated with microfluidics is widely used to measure the pH of samples available in small volumes. Single pass absorption spectroscopy, however, suffers from poor minimum detectable absorption coefficient because the optical path length is determined by the physical dimensions of microchannels. As a result, either large amounts of indicator dyes are added or instrumentally complex techniques are used to determine sample pH. This work addresses these limitations by the integration of metal-clad leaky waveguide (MCLW) with microfluidics to perform broadband absorption spectroscopy. The MCLW consisted of a porous agarose waveguide with an estimated thickness and swelling ratio values of 1.405 ± 0.009 μm and 20.96 ± 0.65% respectively coated onto a titanium layer 32.8 ± 0.43 nm thick on a glass slide. MCLW permits obtaining absorption spectra over most of the wavelength range of the visible region (i.e. 450 nm to 700 nm) with a speed of 12.5 spectra per second using 3.2 nl of sample. The minimum detectable decadic absorption coefficient measured using MCLW was 0.43 cm⁻¹, which is intermediate between single pass absorption spectroscopy and instrumentally complex techniques such as broadband cavity enhanced absorption spectroscopy and whispering gallery mode resonators.

We report a novel self-referenced diffraction-based leaky waveguide (LW) comprising a thin (~2 µm) film of a photofunctionalisable hydrogel created by covalent attachment of a biotinylated photocleavable linker to chitosan. Streptavidin attached to the chitosan via the photocleavable linker was selectively removed by shining 365 nm light through a photomask to create an array of strips with high and low loading of the protein, which served as sensor and reference regions respectively. The differential measurements between sensor and reference regions were used for measuring analytes (i.e., biotin protein A and IgG) while reducing environmental and non-specific effects. These include changes in temperature and sample composition caused by non-adsorbing and adsorbing species, leading to reduction in effects by ~98%, ~99%, and ~97% respectively compared to the absolute measurements. The novelty of this work lies in combining photofunctionalisable hydrogels with diffraction-based LWs for referencing. This is needed to realise the full potential of label-free optical biosensors to measure analyte concentrations in real samples that are complex mixtures, and to allow for sample analysis outside of laboratories where drifts and fluctuations in temperature are observed.