Waveguide-based Chemo-and Biosensors : Complex Emulsions for the Detection of Caffeine and Proteins

We report on a new modular sensing approach in which complex emulsions serve as efficient transducers in optical evanescent field-based sensing devices. Specifically, we leverage the tunable refractive index upon chemically triggered changes in droplet morphology or orientation. Variations in the optical coupling result in readily detectable changes in the light transmitted from a waveguide.


Materials and Methods
All chemicals were purchased from Sigma-Aldrich and were used as received, unless otherwise noted.The emulsion droplet oils, dibutylphthalate and methoxyperfluorobutane, were obtained from Sigma-Aldrich and SynQuest, respectively.The artificial active surfactants for the sensing of caffeine and concanavalin A were synthesized according to the procedures detailed below.DI water was used for the preparation of the continuous phases.
NMR spectra were recorded using a Bruker Avance 400 MHz NMR spectrometer.Chemical shifts (δ) are reported in parts per million (ppm), spectra were referenced with TMS for 1 H NMR. Deuterated solvents were obtained from Cambridge Isotope Laboratories Inc. Mass spectral data were obtained with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS) at the MIT Department of Chemistry Instrumentation Facility.Samples were ionized with the electrospray technique (ESI).ATR-FTIR spectra were obtained using a Thermo Scientific Nicolet 6700 FTIR with a Ge crystal for ATR.Absorption spectra were obtained using an Agilent Cary 4000 UV/Vis spectrophotometer.Bright-field images were taken with a Zeiss Axiovert 200 Inverted Microscope and a Zeiss AxioCam HRc camera.Test experiments with the emulsions were observed on an Amscope Binocular Stereo Microscope.For the optical real-time detection of small morphology changes the complex emulsions were placed onto the hypotenuse side of a UV fused silica right-angle prism, purchased from Thorlabs.Light intensities were recorded using a fiber optic mount.A collimated laser diode module ( = 405 nm; 4.5 mW; Ø: 11 mm; Thorlabs) with a round beam profile was used as the excitation source/light beam.An AvaSpec-ULS2048 StarLine Fiber-optic Spectrometer (Avantes) was used for recording the emission intensities.For the preparation of monodispersed complex emulsion droplets the following equipment was purchased from Dolomite Microfluidics: two Mitos P-Pumps, Basic, and the Remote partnered with an external Remote Chamber 30 mL, Telos® High Throughput Droplet System, Telos 2 Reagent Chip (100µm), 1.6mm O.D. x 0.25mm I.D. FEP tubing, End Fittings and Ferrules, Linear Connector Funnel with FEP Tubing, 1/16" x 0.8mm, 10 meters, and T-Connector.

Fabrication of Complex Emulsions
Emulsification was conducted at temperatures above the critical solution temperature (28 °C) of the dispersed phase solvents, typically a 1:1 mixture of dibutylphthalate and methoxyperfluorobutane.The continuous phase consisted of 1 wt.% solution of SDS and Zonyl-300 in varying ratios.Cooling the emulsion droplets below the critical solution temperature induces phase separation inside the droplets, as described previously. 1 Emulsions were fabricated using a microfluidics device.This procedure allows for the formation of droplets with highly uniform morphology, composition, and size.Emulsion droplets were fabricated using a Dolomite Microfluidic Setup inside a laminar flow hood using a Telos 2 Reagent Chip (100µm).Two Mitos P pressure pumps, one for the mixed droplet phases and one for the continuous phase, were used for controlling the flow rate.After heating the droplet phase above the upper critical solution temperature, the fluids were driven by pressurizing the two individual droplet and continuous chambers with N 2 providing a pulseless, stable flow to the flow focusing chip (pressures: droplet phase: 300 mbar; continuous phase: 320 mbar).The droplet phase was split into two crinkled adjacent flow resistors which provide additional flow stability and mixing.For the fabrication of dyed complex emulsions, perylene (1.5 mM) was dissolved in the hydrocarbon phase prior to emulsification.

Waveguide Read-Out
In order to realize an optical read-out of small morphological changes, as-prepared complex emulsion droplets were placed onto the hypotenuse side of a right-angle glass prism (Thorlabs, borosilicate right angle glass prism; 20mm) .First, 200 L of pure surfactant solution was placed onto the surface of the prism using a micropipette.Then, 20 L of droplets were added into the surfactant solution on the surface.Due to gravity, the monodisperse droplets started spreading and ultimately aligned in a perfect monolayer, with the fluorocarbon phase facing downwards.A collimated laser beam ( = 405 nm; laser diode: Thorlabs) was coupled into the glass prism and directed such that it hits the hypotenuse side of the glass prism (area of the droplets) at an angle of ~ 70°, which is above the critical angle, resulting in TIR of the laser beam at the interface between the glass slide (n g = 1.47) and the aqueous surfactant solution (n W = 1.33) containing the droplets.An optical fiber (NA: 0.22) was mounted in a distance of 2.0 cm above the droplet monolayer.The optical fiber was connected to an AvaSpec-ULS2048 StarLine Fiber-optic Spectrometer (Avantes) for recording the light intensities (integration time 3 msec; averaging 50 spectra).For the determination of the calibration curves (i.e., the emission intensity as a function of the droplet morphology) of dyed emulsion droplets, pre-fabricated monodisperse droplets were placed in surfactant solutions containing different ratios of SDS and Zonyl prior to the deposition on the prism.The recorded emission intensities were normalized to the highest light intensity measured for droplets in the double emulsion state.

Synthesis of Pincer Surfactant
The synthesis of Pd-pincer surfactant C 10 P followed a modified literature procedure: 2

Synthesis of 4-hydroxypyridine-2,6-dicarboxylic acid (2)
Chelidonic acid (1 g, 5.43 mmol, 1 equiv.)was added to an ammonia solution (28-30 %, 11 mL, 25 equiv.)and cooled to 0 °C in an ice bath.The mixture was heated to 100 °C for 6 h, then the solution was cooled to room temperature again.Hydrochloric acid was added carefully until no more precipitation of the product occurred.The solid was filtered, washed with ice water and dried in the vacuum oven at 55 °C overnight to give 2 as a white solid (807 mg, 4.41 mmol, Yield: 82 %).
No signals observed in 1 H NMR spectrum in D 2 O, Chloroform-d or DMSO-d.

Microscopy Read-Out
We visualized the change in morphology of the complex emulsions when caffeine was added using inverted optical microscopy.Similar to the qualitative demonstration using optical scattering, we started with the Janus droplets in a mixture of C 10 P and Zonyl surfactants.We then obtained optical micrographs upon the step-wise additions of caffeine.To produce a quantitative readout from the micrograph, we implemented an image processing program that detects the radius of the droplets (R out ) and the radius of the inner phase (R in ) and calculates the ratio between the two radii (R in /R out ), Figure S3.
At the starting Janus condition, the value of the ratio between the radii is close to unity, Figure S3B.And upon the addition of caffeine, the complex emulsions transform from Janus droplets toward double emulsions (with the organic phase encapsulated), thus reducing the value of the inner phase radius and the ratio, Figure S3C.We chose to measure the ratio between the two radii rather than just the radius of the inner phase to eliminate the variability arising from polydispersity in size of the droplets.Figure S3D shows the gradual decrease in the ratio of the radii with higher concentration of caffeine, as expected when the Janus emulsions transform into double emulsions.

Waveguide Read-Out
For sensing of caffeine, droplets were prepared using the as-synthesized Pd-pincer complex C 10 P as the organic surfactant instead of SDS.Before recording the calibration curves, droplets were added to a surfactant solution containing a 95:5 ratio of C 10 P:Zonyl (overall surfactant concentration 0.1 wt.% and 1.0 wt.%, respectively).The ratio of the surfactants in the aqueous phase (C 10 P and Zonyl) was strategically chosen such that the droplet morphology at the starting point (i.e., no caffeine present) resulted in an almost encapsulated double emulsion state resulting in a close to the maximum emission intensity.Subsequently, for sensing, vials with the respective surfactant solution mixture containing different concentrations of caffeine in an overall volume of 1 mL were prepared.Then 100 L of the prepepared complex emulsions were added.The emulsions were agitated in a Labnet Vortemp 56 incubator (shaking speed of 150 rpm at rt) for 10 min prior to measuring the emission intensities as described above.In a typical setup we used 1 wt.% surfactant concentrations, which allows for the precise detection of caffeine concentrations below 0.5 mM, a concentration regime of interest for the monitoring of caffeine content in coffee.The decrease of the surfactant effectiveness and thus a decrease in the interfacial tensions resulted in a step-wise decrease of the light intensity measured above the droplet monolayer.At low concentrations, the calibration curve is relatively linear and provides precise detection of caffeine (R 2 =0.99).A decrease of the surfactant concentrations from 1 wt.% down to 0.1 wt.% results in an increase of sensitivity towards the detection of caffeine in the concentration regime between 0 and 50 M (R 2 =0.98).The procedure was repeated five times for each caffeine concentration.

Sensing of ConA
In order to monitor the emission intensity upon droplet agglutination, we first prepared droplets using a mannose surfactant, as reported previously. 3In brief, for the continuous phase, ManC14 and Zonyl FS-300 were used to stabilize and generate emulsions with Janus morphology.The two surfactants were dissolved in HEPES buffer solution (10 mM, containing 1 mM CaCl 2 , and 1 mM MnCl 2 , pH = 7.5) separately with concentration of 0.01% by weight.The final volume ratio between ManC14 solution and Zonyl FS-300 solution was kept at 3:1 to generate Janus emulsions.The Janus emulsions were subsequently added to different vials with the same surfactant solution but containing different concentrations of concanavalin A (ConA).The solutions were then swirled gently in a Labnet Vortemp 56 incubator (shaking speed of 150 rpm at rt) for 6 h.Then, the droplets were deposited onto the prism and the light intensities were recorded as described above.

Figure S1 :
Figure S1: Qualitative demonstration of the working principle of the pincer complex based on the lens effect.Transmission changes upon change in the morphology of the droplets.(A) Schematic drawing of the morphology of the droplets and their transmission.(B) Images of the droplets in double emulsion and Janus state.(C) Change of transmission from double emulsion to Janus state morphology in droplet solution.The Logo underneath the sample plate only becomes visible if droplets are in Janus morphology and transmissive.

Figure S2 :
Figure S2: Analysis of the optical micrographs of the changes in droplet morphology upon the addition of caffeine.(A) Side view and Top view schematic diagrams of the droplets in Janus and double emulsion states, depicting the radius of the droplet (R out ) and the inner radius (R in ).The ratio of the radii is near unity in Janus emulsions and approaches 0.8 for double emulsions.Top view optical micrographs of Janus emulsions with C 10 P and Zonyl surfactants (B) and double emulsions after the addition of 1 mM of caffeine (C).(D) The ratio between R in and R out as a function of the concentration of caffeine.