Microgel assisted Lab-on-Fiber Optrode

Precision medicine is continuously demanding for novel point of care systems, potentially exploitable also for in-vivo analysis. Biosensing probes based on Lab-On-Fiber Technology have been recently developed to meet these challenges. However, devices exploiting standard label-free approaches (based on ligand/target molecule interaction) suffer from low sensitivity in all cases where the detection of small molecules at low concentrations is needed. Here we report on a platform developed through the combination of Lab-On-Fiber probes with microgels, which are directly integrated onto the resonant plasmonic nanostructure realized on the fiber tip. In response to binding events, the microgel network concentrates the target molecule and amplifies the optical response, leading to remarkable sensitivity enhancement. Moreover, by acting on the microgel degrees of freedom such as concentration and operating temperature, it is possible to control the limit of detection, tune the working range as well as the response time of the probe. These unique characteristics pave the way for advanced label-free biosensing platforms, suitably reconfigurable depending on the specific application.


S1. MGs functionalization
5 mL of purified P(NIPAM-AAc) MGs was cooled at 4°C and added to a solution of 250mM of EDC and 125mM APBA in pH 4.7 MES buffered saline. The solution was cooled at 4°C, and the reaction mixture was allowed to proceed overnight at 4°C. The products were purified by dialysis against water. Negative control samples were similarly prepared by using P(NIPAM-AAc) MGs reacted with APBA only (without EDC) in pH 4.7 MES buffer.

S2. Numerical simulations
For the numerical analysis we used the commercial software COMSOL Multiphysics (RF module) based on the finite element method [1]. Following a similar approach of ref. [2], by exploiting the translational symmetries, the computational domain has been reduced to the unit cell corresponding to the lattice period, terminated with Floquet periodic boundary conditions, placed two-by-two in the opposite walls.
The resulting structure supports a transverse electromagnetic (TEM) wave emulating the normally incident plane wave; we have used this kind of excitation instead of a proper fiber mode with Gaussian profile to simplify our simulations. Gold and silica have been modeled with dispersive refractive index functions taken respectively from [3] and [4]. In Fig. S2.1, the cross section view of the computational domain is shown. In the wake of our AFM morphological measurements, we have considered the patterned hole sidewalls tilted of an angle γ1 with respect to fiber tip plane.
With reference to Fig. S2.1, the gold and chromium layers thickness are respectively hg and hc, while d is the hole diameter evaluated at half of total metallic layer thickness (hg+ hc). A conformal dielectric layer on the metallic nanostructure has been considered in our model, with the possibility to control its thickness hd and its hole sidewall inclination. By taking into account fabrication tolerances, the structure has been modeled by setting a=650nm, FF=0.3, d=2•FF•a=390nm, hg=33nm, hc=5nm, γ1= γ2=30°. Sensitivities of the standard EOT configuration (i.e. the structure without MGs deposition) have been evaluated. In that case, the dielectric layer mimics the presence of a bio-molecules on the substrate, and has been modeled considering a refractive index equal to 1.45. In order to evaluate the surface sensitivity we considered the resonance minimum position as function of biological layer thickness, varying in the range between 5nm and 30nm, end extracting the interpolation line slope (  With the assumption that the MGs deposited on the nanostructure constitutes a uniform layer with an average refractive index, the effect of the MGs swelling followed by the consequential refractive index change have been simulated keeping fixed the structure morphological parameters.    Eppendorf tube holder heated by Peltier cells control system. Specifically the source is a supercontinuum lasers with its collimator inserted into a splitter that provides two outputs with different wavelength ranges. The IR output (800-2400 nm) is connected to a fiber collimator through a coupler equipped by alignment mirrors. With reference to Fig. S3.1, a first isolator is placed in output to source, while the other two are used just before the two OSA in order to avoid spurious reflected components coupled in the optical circuit. The reflected spectrum is given by the ratio between the data acquired from OSA1 and OSA2 respectively, normalized with respect to the transfer functions of the paths AC, and ADB of the coupler. The normalization allows to have a spectrum not influenced by the paths asymmetry and by both the coupler and isolators dispersive behavior. A Labview plug-in as been used for the OSA scan timing control (via GPIB interface protocol), ensuring the acquisitions simultaneity, and consequently improving the robustness against the source undesired oscillations.

S4. DLS measurements
Measurements were conducted using a DLS system (Malvern Zetasizer Nano ZS instrument, 633 nm laser, 173° scattering angle) equipped with a temperature controller. For thermo-responsivity measurements, an equilibration time of 1200s was used for each temperature and a total of 5 run

S5. Description of the chamber for thermal measurements
In the Fig. S5 is shown the Eppendorf tube holder enabling the temperature control of the experimental environment.
It is composed of a brass die machined with a CNC mill that match perfectly the Eppendorf tube  We carried out glucose detection experiments by using the LOF probe without MGs integration.
This test is a sort of reference and it is useful for trying to correctly evaluate the sensitivity enhancement due to the integration of the MGs layer. To this aim, in order to make the LOF device receptive to glucose molecules, we adopted the same functionalization procedure used to  We also experimentally evaluated the sensorgrams relative to the device integrated with functionalized MGs but not activated by EDC (negative control); Also in this case, as shown in figure S6.2, without the boronic acid acting as ligand, no wavelength shift is observed.

S7 Glucose assay kit protocol (enzymatic colorimetric assay)
The coupling of APBA on gold surface and its capability to bind glucose molecules was evaluated by using a Glucose (GO) Assay Kit purchased from Sigma Aldrich. The colorimetric assay involved the following steps: i) glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase.; ii) hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to form a colored product; iii) oxidized o-dianisidine reacts with sulfuric acid to form a stable colored product (pink color).
The intensity of the pink color is proportional to the original glucose concentration.
To perform the colorimetric assay, the optical fiber probes was dipped in a standard glucose solution [1mg/ml] for 30 minutes. The probes were then placed in a tube containing 5µL of carbonate buffer and 10µL of Assay Reagent for 30 minutes at 37 °C. The reaction was stopped by adding 10µL of 12N H2SO4 into each tube. The probe functionalized with APBA showed a light pink color that confirmed the presence of glucose molecules bound to the boronic acid moieties. For the negative control, the pink color production was not observed: in absence of APBA glucose molecules are not able to bind to gold surface.