Ultra-stable liquid crystal droplets coated by sustainable plant-based materials for optical sensing of chemical and biological analytes

Herein, we demonstrate for the first time the synthesis of ultra-stable, spherical, nematic liquid crystal (LC) droplets of narrow size polydispersity coated by sustainable, biodegradable, plant-based materials that trigger a typical bipolar-to-radial configurational transition in dynamic response to chemical and biological analytes. Specifically, a highly soluble polymer, potato protein (PoP) and a physically-crosslinked potato protein microgel (PoPM) of ∼100 nm in diameter, prepared from the PoP, a byproduct of the starch industry, were compared for their ability to coat LC droplets. Although both PoP and PoPM were capable of reducing the interfacial tension between water and n-tetradecane <30 mN m−1, PoPM-coated LC droplets showed better stability than the PoP-coated droplets via a Pickering-like mechanism. Strikingly, the Pickering LC droplets outperformed PoP-stabilized droplets in terms of dynamic response with 5× lower detection limit to model chemical analytes (sodium dodecyl sulphate, SDS) due to the difference in SDS-binding features between the protein and the microgel. To eliminate the effect of size polydispersity on the response, monodisperse Pickering LC droplets of diameter ∼16 μm were additionally obtained using microfluidics, which mirrored the response to chemical as well as biological analytes, i.e., primary bile acid, an important biomarker of liver diseases. We demonstrate that these eco-friendly microgels used for creating monodisperse, ultra-stable, LC complex colloids are powerful templates for fabricating next generation, sustainable optical sensors for early diagnosis in clinical settings and other sensing applications.

For fabricating the aqueous dispersion of potato protein microgels (PoPM), a slightly modified top-to-down approach described by Sarkar et al. was used. 2 For this, 10.0 wt% PoP solution was heated in temperature controlled water bath at 80 °C for 30 min to denature patatins. The resultant heat-set PoP-based hydrogel was cooled down to 25 °C and stored at 4 °C overnight. To obtain the PoPM, firstly, the hydrogel was mixed with Milli Q water (1:1 w/w) and prehomogenized using a hand blender (HB711M, Kenwood, UK) to create macrogel particles. The 50 vol% PoP macrogel particle containing 5.0 wt% PoP was degassed using a Thinky instrument (Intertronics, Thinky ARE-250, Oxfordshire, UK). Finally, the defoamed dispersion of PoP macrogel particle was homogenized by passing through a two-stage valve homogenizer (Panda Plus 2000, GEA Niro Soavi Homogeneizador, Parma, Italy) four times operating at first/second stage pressures of 200/100 bars, respectively. The resultant solution was termed as 5.0 wt% PoPM aqueous solution, i.e., microgel particles containing 5.0 wt% protein, with a volume fraction of 50 vol% PoPM, which was diluted with Milli Q water at pH 6.15 to create various concentrations of the microgels for fabricating Pickering LC emulsions.

Mesh size of PoPM.
The mesh size of the PoPM particles was calculated indirectly by considering that PoPM is an average nanometric unit of the PoP-hydrogel, and no syneresis occurred during the formation of the microgels. 3 The average mesh size, ξ, is related to the storage modulus, G', obtained for the PoP hydrogel as given by the following equation: 4,5 (1) where, K B and T are Boltzmann constant and temperature, respectively.
To measure G', the thermal gelation of PoP in the rheometer was followed by a frequency sweep test of the PoP gel. For this, a modular compact rheometer, MCR 302 (AntonPaar, Graz, Austria) equipped with a cone and plate (CP 50, 50 mm diameter and cone angle 2°) geometry was used. For gelation, 10.0 wt% PoP aqueous solution was placed in the measuring cell carefully to avoid bubbles formation. The silicone oil and adiabatic hood were used to seal the cell to prevent evaporation. Firstly, the cell temperature was increased from 25 °C to 80 °C, followed by keeping the cell at 80 °C for 10 min. After PoP gelation, the cell temperature was reduced to 25 °C before the frequency sweep test was performed with varying angular frequency, ω from 1 to 100 rad/s keeping the shear strain constant at 0.1 %. The G' and loss modulus, G" for PoP-gel were obtained where the final G' value at 100 rad/s was used to calculate mesh size.

Circular dichroism.
To understand the heat-induced conformational change in the secondary and tertiary structure of PoP while forming PoPM, the far and near UV circular dichroism (CD) spectra of PoP solution and PoPM dispersion were recorded, respectively, using Chirascan Plus, Applied photophysics Spectropolarimeter (Leatherhead, UK). For analysis, 0.025 wt% of PoP solution and PoPM dispersion were prepared using Milli Q water as solvent. For far UV spectra, the measurements were performed using a 1 mm path length cell at 180 -260 nm, while for near UV spectra, a 10 mm path length cell at 240 -350 nm was used. Both analyses were carried out using quartz cuvettes and with the temperature maintained at 20 °C, 2 nm bandwidth and 1 nm step size. The obtained spectra were corrected by subtracting Milli Q water as the baseline.   objective and DeltaPix Invenio 3SII camera (Smørum, Denmark). 100 different bright field images of each emulsion were taken and analysed using the ImageJ software to obtain the diameter of the individual E7 droplets. The droplet size distribution and polydispersity of emulsions were reported in terms of Sauter mean diameter, D [3,2] , and De Broucker mean diameter, D [4,3] , and PDI, respectively, using equations 2, 3 and 4:

Dynamic interfacial tension measurements. For comparison of interfacial properties of
(2) [3,2] where, N i is the number of droplets with diameter D i , σ is the standard deviation in obtained diameter data, and D av is the average mean droplet diameter. were carefully collected using a syringe, and their absorbance was recorded using a UV-Vis Spectrophotometer (Multiskan FC microplate photometer, Thermo Scientific) at 595 nm using a standard Bradford assay kit. The protein concentrations in the supernatants were determined using the Lowry method with Bovine serum albumin (BSA) as a standard. The adsorption efficiency, α, defined as the percentage of emulsifier (PoP and PoPM) adsorbed to the LC droplets relative to the total added content and the effective adsorption density, Γ ads , i.e., the density of emulsifier at the interface, were evaluated using equation (5) and (6), respectively as reported elsewhere. 3 (5) = × 100 [3,2] where, c adsorbed and c initial are the concentration of the adsorbed and initially added emulsifier (PoP and PoPM) in the emulsion, respectively with c adsorbed = c initial -c unadsorbed . ϕ is the volume fraction of the LC phase, and D [3,2] is the Sauter mean diameter of the LC droplets.

ζ-potential.
The ζ-potential of the protein, microgel, and the E7-in-water emulsions with or without analytes was evaluated using Zetasizer Nano-ZS (Malvern Instruments Ltd., Worcestershire, UK) instrument operating at a detection angle of 173° and with 633 nm He-Ne laser light source using folded capillary cell DTS 1070. For PoP and PoPM, the stock solutions were diluted to 0.01 wt% protein concentration, and for emulsions, all stabilized emulsions were diluted ten-fold while the bare emulsion was diluted five-fold prior to measurements. In the presence of analytes, the emulsions were incubated with analytes for 30 min before the ζ-potential measurement.

Statistical analyses.
All measurements were performed three times on triplicate samples and reported as means and standard deviations (n = 3 × 3). The statistical analyses were conducted using one-way (ANOVA), and samples were considered to be significantly different with p < 0.05 using the Tukey test.  *Note. In the first study, the emulsions were analyzed after incubation with the analytes for 30 min. For the second study with SDS and NaCh, firstly, the emulsion was incubated with SDS for 30 min., and then NaCh was added. The measurements were made after 30 min of NaCh addition. Here, all the emulsions were diluted ten folds for the measurement.