Lab-on-chip fabrication and functionalization

The Mid-IR nanoantenna sensor was realized by Electron Beam Lithography (EBL) direct writing on 1-mm thick, optical grade CaF₂ substrate (by Crysel). The antenna array with nm-size precision was patterned on a 300 nm thick Poly(methyl methacrylate) (PMMA) EBL resist layer. To prevent resist and substrate charging during the e-beam exposure, a phenomenon leading to deformations of the nanoscale antenna definition, the PMMA resist layer was coated with a thin (5 nm) Cr layer. Such charge compensation layer was then removed right after the exposure and before the resist developer, via wet etching. The final dipolar antennas are realized via deposition and liftoff of 10/80 nm Cr/Au metal layers. The optimized double-band antenna array was implemented by realizing and testing a set of samples with different array sizes (rod length, width, and spacing), then tuning the final design to precisely match the desired antenna resonances. Gold nanoantennas of different lengths in Fig. 2 were realized by applying a scaling factor to the original CAD 2D design of ± 10% and ± 5%.

Gold nanoantennas were functionalized according to Liu et al.[35]. Briefly, a 10 mM mixture of polyethylene glycol (PEG) molecules of two different molecular weights (3:1 molar ratio) was prepared in DPBS [113]. For this purpose, we used a short PEG chain (Methyl-PEG-Thiol MT(PEG)₄, MW 200 kD, ThermoFisher Scientific) and a long PEG chain, namely biotinylated-polyethylene glycol-thiol (mPEG-Biotin, MW 1000 kD Nanocs). After PEG binding to the gold nanostructures, unbound PEG molecules were washed in DPBS, and the long PEG biotinylated chains were conjugated with Neutravidin (NeutrAvidin Biotin Binding, ThermoFisher). For this purpose, a 0.05 mg/mL neutravidin solution was prepared in DPBS [114, 115]. The surface of the device was further functionalized for exosome immunocapture using a 0.05 mg/mL Anti-CD63 biotinylated antibody [Anti-CD63 (MEM-259) BIOTIN ab134331, abcam] solution in DPBS and exploiting the high affinity of the multiple neutravidin binding sites for biotin.

Before measurements, the patterned CaF₂ widow was arranged in a 3D-printed wet sample holder fabricated as follows. A 3D CAD of the device, consisting of three different components, was created with Rhinoceros®, then exported as a stl file to CURA software which created the G-Code script containing the commands to guide the printing process. The fabrication was realized by Fusion Deposition Modelling (FDM) employing an Ultimaker S3 printer and a Ultimaker Black Tough PLA filament (section of 2.85 mm). The filament was extruded at 210 °C, using a 0.25 mm diameter nozzle, and deposited on a heated build plate (65 °C). The devices were printed at 25 mm/s speed, taking about 8 h for the realization of the three components. The structures were printed layer by layer, each one with a 0.1 mm thickness, a line width of 0.23 mm, and a grid infill pattern at a density of 70%.